Activation of HCV-specific T cells

ABSTRACT

The invention provides a method of activating hepatitis C virus (HCV)-specific T cells, including CD4 +  and CD8 +  T cells. HCV-specific T cells are activated using fusion proteins comprising HCV NS3, NS4, NS5 a , and NS5 b  polypeptides, polynucleotides encoding such fusion proteins, or polypeptide or polynucleotide compositions containing the individual components of these fusions. The method can be used in model systems to develop HCV-specific immunogenic compositions, as well as to immunize a mammal against HCV.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/281,341, filed Oct. 25, 2002, which is a continuation-in-part of U.S.application Ser. No. 09/698,874, filed Oct. 27, 2000, from whichapplications priority is claimed under 35 USC §120. U.S. applicationSer. No. 09/698,874 claims the benefit of provisional patent applicationSer. No. 60/161,713, filed Oct. 27, 1999 under 35 USC §119(e)(1). Theforegoing applications are incorporated herein by reference in theirentireties.

TECHNICAL AREA OF THE INVENTION

The invention relates to the activation of hepatitis Cvirus(HCV)-specific T cells. More particularly, the invention relates tothe use of multiple HCV polypeptides, either alone or as fusions, tostimulate cell-mediated immune responses, such as to activateHCV-specific T cells.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is an important health problem withapproximately 1% of the world's population infected with the virus. Over75% of acutely infected individuals eventually progress to a chroniccarrier state that can result in cirrhosis, liver failure, andhepatocellular carcinoma. See Alter et al. (1992) N. Engl. J. Med.327:1899-1905; Resnick and Koff. (1993) Arch. Intem. Med. 153:1672-1677;Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al. (1995) N. Engl. J.Med. 332:1463-1466.

Despite extensive advances in the development of pharmaceuticals againstcertain viruses like HIV, control of acute and chronic HCV infection hashad limited success (Hoofnagle and di Bisceglie (1997) N. Engl. J. Med.336:347-356). In particular, generation of a strong cytotoxic Tlymphocyte (CTL) response is thought to be important for the control anderadication of HCV infections. Thus, there is a need in the art foreffective methods of inducing strong CTL responses against HCV.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods forstimulating immune responses, such as activating T cells which recognizeepitopes of HCV polypeptides. This and other objects of the inventionare provided by one or more of the embodiments described below.

The invention provides HCV proteins useful for stimulating immuneresponses, such as activating HCV-specific T cells. One embodimentprovides a fusion protein that comprises HCV polypeptides, wherein theHCV polypeptides consist essentially of an NS3, an NS4, an NS5apolypeptide, and optionally a core polypeptide. In certain embodiments,the fusion protein includes an NS5b polypeptide.

In certain embodiments, at least one of the HCV polypeptides is derivedfrom a different strain of HCV than the other polypeptides.

The invention also provides compositions comprising any of these fusionproteins and a pharmaceutically acceptable excipient. In certainembodiments, the compositions further comprise an adjuvant, a CpGpolynucleotide and/or the fusion protein is adsorbed to or entrappedwithin a microparticle or ISCOM. The compositions can further comprise apolynucleotide encoding an E1E2 complex. The E1E2 polynucleotide canalso be adsorbed to or entrapped withing a microparticle.

Another embodiment provides a composition comprising HCV polypeptidesand a pharmaceutically acceptable excipient. The HCV polypeptidesconsist essentially of an NS3, an NS4, an NS5a polypeptide, andoptionally a core polypeptide. In certain embodiments, the compositionincludes an NS5b polypeptide. In other embodiments, the compositionsfurther comprise an adjuvant, a CpG polynucleotide and/or one or more ofthe HCV polypeptides is adsorbed to or entrapped within a microparticleor ISCOM. The compositions can further comprise a polynucleotideencoding an E1E2 complex. The E1E2 polynucleotide can also be adsorbedto or entrapped withing a microparticle. Moreover, one of the HCVpolypeptides may be derived from a different strain of HCV than theothers.

Even another embodiment of the invention provides an isolated andpurified polynucleotide which encodes a fusion protein as describedabove. In additional embodiments, the fusion proteins further include apolynucleotide encoding an E1E2 complex.

Yet another embodiment of the invention provides a compositioncomprising the polynucleotides described above and a pharmaceuticallyacceptable excipient. In certain embodiments, the compositions furthercomprise an adjuvant and/or the polynucleotide may be adsorbed to orentrapped within a microparticle. The compositions can further comprisea polynucleotide encoding an E1E2 complex. The E1E2 polynucleotide canalso be adsorbed to or entrapped withing a microparticle.

In a further embodiment, the invention provides a composition comprisingHCV polynucleotides and a pharmaceutically acceptable excipient, whereinthe HCV polynucleotides consist essentially of polynucleotides encodingan NS3, an NS4, an NS5a polypeptide, and optionally a core polypeptide.In certain embodiments, the composition also includes a polynucleotideencoding an NS5b polypeptide. The compositions may further comprise anadjuvant and/or one or more of the polynucleotides may be adsorbed to orentrapped within a microparticle. The compositions can further comprisea polynucleotide encoding an E1E2 complex. The E1E2 polynucleotide canalso be adsorbed to or entrapped withing a microparticle. Additionally,one or more of the polynucleotides may be derived from a differentstrain of HCV than the others.

In another embodiment, the invention provides a method of activating Tcells which recognize an epitope of an HCV polypeptide. T cells arecontacted with any of the fusions, polynucleotides or compositionsdescribed above. A population of activated T cells recognizes an epitopeof the NS3, NS4, NS5a, NS5b, core and/or E1E2 polypeptide.

In the proteins and polynucleotides above, the regions in the fusionsneed not be in the order in which they naturally occur in the native HCVpolyprotein. Thus, for example, the NS5b polypeptide, if present, may beat the N- and/or C-terminus of the fusion, or may be located internally.Similarly, the E1 polypeptide may precede or follow the E2 polypeptide.The E1E2 polypeptide may also be part of the nonstructural fusionprotein or may be provided separately, as an E1E2 complex, or asindividual polypeptides.

Moreover, the NS3 polypeptide may include a modification to inhibitprotease activity, such that cleavage of the fusion is inhibited. Suchmodifications are described more fully below. Additionally, thecompositions can comprise more than one HCV nonstructural fusionprotein, such as a fusion protein with NS3, NS4 and NS5a, and a fusionprotein with NS3, NS4, NS5a, NS5b and E1E2. The E1E2 complexes, whetherpresent separately or as part of the fusion, can have varying E1E2polypeptides (described more fully below).

In certain embodiments, the nonstructural fusion protein consists of,from the amino terminus to the carboxyl terminus, an NS3, an NS4, anNS5a and, optionally, an NS5b polypeptide and the E1E2 complex consistsof, from amino terminus to the carboxyl terminus, an E1 polypeptide andan E2 polypeptide.

The various polypeptides (and polynucleotides encoding therefor) arederived from the same HCV isolate, or from different strains andisolates including isolates having any of the various HCV genotypes, toprovide increased protection against a broad range of HCV genotypes.

Yet another embodiment of the invention provides a method of stimulatingan immune response, such as a cellular immune response, in a vertebratesubject by administering a composition as described herein. In certainembodiments, the composition activates T cells which recognize anepitope of an HCV polypeptide. T cells are contacted with a compositionas described above. A population of activated T cells recognizes anepitope of one or more of the HCV polypeptide(s).

The invention thus provides methods and reagents for stimulating immuneresponses to HCV, such as for activating T cells which recognizeepitopes of HCV polypeptides. These methods and reagents areparticularly advantageous for identifying epitopes of HCV polypeptidesassociated with a strong CTL response and for immunizing mammals,including humans, against HCV.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of the HCV genome, depicting thevarious regions of the HCV polyprotein.

FIG. 2 depicts the DNA and corresponding amino acid sequence of arepresentative native NS3 protease domain.

FIGS. 3A-3C (SEQ ID NOS:3 and 4) shows the nucleotide and correspondingamino acid sequence for the HCV-1 E1/E2/p7 region. The numbers shown inthe figure are relative to the full-length HCV-1 polyprotein. The E1, E2and p7 regions are shown.

FIG. 4 is a diagram of plasmid pMHE1E2-809, encoding E1E2₈₀₉, arepresentative E1E2 protein for use with the present invention.

FIGS. 5A-5J (SEQ ID NOS:7 and 8) depict the DNA and corresponding aminoacid sequence of a representative NS345Core fusion protein. The depictedsequence includes amino acids 1242-3011 of the HCV polyprotein(representing polypeptides from NS3, NS4, NS5a and NS5b) with aminoacids. 1-121 of the HCV polyprotein (representing a polypeptide from thecore region) fused to the C-terminus of NS5b. This numbering is relativeto the HCV-1 polyprotein.

FIG. 6 shows a side-by-side comparison of IFN-γ expression generated inanimals in response to delivery of alphavirus constructs encodingNS3NS4NS5a.

FIG. 7 shows IFN-γ expression generated in animals in response todelivery of plasmid DNA encoding NS3NS4NS5a (“naked”), PLG-linked DNAencoding NS3NS4NS5a (“PLG), separate DNA plasmids encoding NS5a, NS34a,and NS4ab (“naked”), and PLG-linked DNA encoding NS5a, NS34a, and NS4ab(“PLG”).

FIG. 8 shows HCV-specific CD8+ and CD4+ responses in vaccinatedchimpanzees.

FIG. 9 depicts the specificity of T cell responses primed byelectroporation of plasmid DNA two weeks subsequent to the thirdimmunization.

FIG. 10 shows the specificity of T cell responses primed by vaccinatingchimpanzees with NS345Core₁₂₁-ISCOMS two weeks subsequent to the thirdimmunization.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (2nd Edition); Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); DNACloning, Vols. I and II (D. N. Glover ed.); Oligonucleotide Synthesis M.J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames & S. J. Higginseds.); Animal Cell Culture (R. K. Freshney ed.); Perbal, B., A PracticalGuide to Molecular Cloning.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an antigen” includes a mixture of two or more antigens,and the like.

The following amino acid abbreviations are used throughout the text:

-   -   Alanine: Ala (A) Arginine: Arg (R)    -   Asparagine: Asn (N) Aspartic acid: Asp (D)    -   Cysteine: Cys (C) Glutamine: Gln (Q)    -   Glutamic acid: Glu (E) Glycine: Gly (G)    -   Histidine: His (H) Isoleucine: Ile (I)    -   Leucine: Leu (L) Lysine: Lys (K)    -   Methionine: Met (M) Phenylalanine: Phe (F)    -   Proline: Pro (P) Serine: Ser (S)    -   Threonine: Thr (T) Tryptophan: Trp (W)    -   Tyrosine: Tyr (Y) Valine: Val (V)

I. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

An HCV polypeptide is a polypeptide, as defined above, derived from theHCV polyprotein. The polypeptide need not be physically derived fromHCV, but may be synthetically or recombinantly produced. Moreover, thepolypeptide may be derived from any of the various HCV strains andisolates including isolates having any of the 6 genotypes of HCVdescribed in Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399 (e.g.,strains 1, 2, 3, 4 etc.), as well as newly identified isolates, andsubtypes of these isolates, such as HCV1a, HCV1b, etc. A number ofconserved and variable regions are known between these strains and, ingeneral, the amino acid sequences of epitopes derived from these regionswill have a high degree of sequence homology, e.g., amino acid sequencehomology of more than 30%, preferably more than 40%, when the twosequences are aligned. Thus, for example, the term “NS4” polypeptiderefers to native NS4 from any of the various HCV strains, as well as NS4analogs, muteins and immunogenic fragments, as defined further below.

By an “E1 polypeptide” is meant a molecule derived from an HCV E1region. The mature E1 region of HCV-1 begins at approximately amino acid192 of the polyprotein and continues to approximately amino acid 383,numbered relative to the full-length HCV-1 polyprotein. (See, FIGS. 1and 3A-3C. Amino acids 192-383 of FIGS. 3A-3C correspond to amino acidpositions 20-211 of SEQ ID NO:4.) Amino acids at around 173 throughapproximately 191 (amino acids 1-19 of SEQ ID NO: 4) serve as a signalsequence for E1. Thus, by an “E1 polypeptide” is meant either aprecursor E1 protein, including the signal sequence, or a mature E1polypeptide which lacks this sequence, or even an E1 polypeptide with aheterologous signal sequence. The E1 polypeptide includes a C-terminalmembrane anchor sequence which occurs at approximately amino acidpositions 360-383 (see, International Publication No. WO 96/04301,published Feb. 15, 1996). An E1 polypeptide, as defined herein, may ormay not include the C-terminal anchor sequence or portions thereof.

By an “E2 polypeptide” is meant a molecule derived from an HCV E2region. The mature E2 region of HCV-1 begins at approximately amino acid383-385, numbered relative to the full-length HCV-1 polyprotein. (See,FIGS. 1 and 3A-3C. Amino acids 383-385 of FIGS. 3A-3C correspond toamino acid-positions 211-213 of SEQ ID NO:4.) A signal peptide begins atapproximately amino acid 364 of the polyprotein. Thus, by an “E2polypeptide” is meant either a precursor E2 protein, including thesignal sequence, or a mature E2 polypeptide which lacks this sequence,or even an E2 polypeptide with a heterologous signal sequence. The E2polypeptide includes a C-terminal membrane anchor sequence which occursat approximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073). An E2 polypeptide, as defined herein, may or may notinclude the C-terminal anchor sequence or portions thereof. Moreover, anE2 polypeptide may also include all or a portion of the p7 region whichoccurs immediately adjacent to the C-terminus of E2. As shown in FIGS. 1and 3A-3C, the p7 region is found at positions 747-809, numberedrelative to the full-length HCV-1 polyprotein (amino acid positions575-637 of SEQ ID NO:4). Additionally, it is known that multiple speciesof HCV E2 exist (Spaete et al., Virol. (1992) 188:819-830; Selby et al.,J. Virol. (1996) 70:5177-5182; Grakoui et al., J. Virol. (1993)67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026). Accordingly,for purposes of the present invention, the term “E2” encompasses any ofthese species of E2 including, without limitation, species that havedeletions of 1-20 or more of the amino acids from the N-terminus of theE2, such as, e.g, deletions of 1, 2, 3, 4, 5 . . . 10 . . . 15, 16, 17,18, 19 . . . etc. amino acids. Such E2 species include those beginningat amino acid 387, amino acid 402, amino acid 403, etc.

Representative E1 and E2 regions from HCV-1 are shown in FIGS. 3A-3C andSEQ ID NO:4. For purposes of the present invention, the E1 and E2regions are defined with respect to the amino acid number of thepolyprotein encoded by the genome of HCV-1, with the initiatormethionine being designated position 1. See, e.g., Choo et al., Proc.Natl. Acad. Sci. USA (1991) 88:2451-2455. However, it should be notedthat the term an “E1 polypeptide” or an “E2 polypeptide” as used hereinis not limited to the HCV-1 sequence. In this regard, the correspondingE1 or E2 regions in other HCV isolates can be readily determined byaligning sequences from the isolates in a manner that brings thesequences into maximum alignment. This can be performed with any of anumber of computer software packages, such as ALIGN 1.0, available fromthe University of Virginia, Department of Biochemistry (Attn: Dr.William R. Pearson). See, Pearson et al., Proc. Natl. Acad. Sci. USA(1988) 85:2444-2448.

Furthermore, an “E1 polypeptide” or an “E2 polypeptide” as definedherein is not limited to a polypeptide having the exact sequencedepicted in the Figures. Indeed, the HCV genome is in a state ofconstant flux in vivo and contains several variable domains whichexhibit relatively high degrees of variability between isolates. Anumber of conserved and variable regions are known between these strainsand, in general, the amino acid sequences of epitopes derived from theseregions will have a high degree of sequence homology, e.g., amino acidsequence homology of more than 30%, preferably more than 40%, more than60%, and even more than 80-90% homology, when the two sequences arealigned. It is readily apparent that the terms encompass E1 and E2polypeptides from any of the various HCV strains and isolates includingisolates having any of the 6 genotypes of HCV described in Simmonds etal., J. Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.),as well as newly identified isolates, and subtypes of these isolates,such as HCV1a, HCV1b etc.

Thus, for example, the term “E1” or “E2” polypeptide refers to native E1or E2 sequences from any of the various HCV strains, as well as analogs,muteins and immunogenic fragments, as defined further below. Thecomplete genotypes of many of these strains are known. See, e.g., U.S.Pat. No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799.

Additionally, the terms “E1 polypeptide” and “E2 polypeptide” encompassproteins which include modifications to the native sequence, such asinternal deletions, additions and substitutions (generally conservativein nature). These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. All of these modifications areencompassed in the present invention so long as the modified E1 and E2polypeptides function for their intended purpose. Thus, for example, ifthe E1 and/or E2 polypeptides are to be used in vaccine compositions,the modifications must be such that immunological activity (i.e., theability to elicit a humoral or cellular immune response to thepolypeptide) is not lost.

By “E1E2” complex is meant a protein containing at least one E1polypeptide and at least one E2 polypeptide, as described above. Such acomplex may also include all or a portion of the p7 region which occursimmediately adjacent to the C-terminus of E2. As shown in FIGS. 1 and3A-3C, the p7 region is found at positions 747-809, numbered relative tothe full-length HCV-1 polyprotein (amino acid positions 575-637 of SEQID NO:4). A representative E1E2 complex which includes the p7 protein istermed “E1E2₈₀₉” herein.

The mode of association of E1 and E2 in an E1E2 complex is immaterial.The E1 and E2 polypeptides may be associated through non-covalentinteractions such as through electrostatic forces, or by covalent bonds.For example, the E1E2 polypeptides of the present invention may be inthe form of a fusion protein which includes an immunogenic E1polypeptide and an immunogenic E2 polypeptide, as defined above. Thefusion may be expressed from a polynucleotide encoding an E1E2 chimera.Alternatively, E1E2 complexes may form spontaneously simply by mixing E1and E2 proteins which have been produced individually. Similarly, whenco-expressed and secreted into media, the E1 and E2 proteins can form acomplex spontaneously. Thus, the term encompasses E1E2 complexes (alsocalled aggregates) that spontaneously form upon purification of E1and/or E2. Such aggregates may include one or more E1 monomers inassociation with one or more E2 monomers. The number of E1 and E2monomers present need not be equal so long as at least one E1 monomerand one E2 monomer are present. Detection of the presence of an E1E2complex is readily determined using standard protein detectiontechniques such as polyacrylamide gel electrophoresis and immunologicaltechniques such as immunoprecipitation.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, or fragments of such derivatives, that retaindesired activity, such as the ability to stimulate a cell-mediatedimmune response, as defined below. In general, the term “analog” refersto compounds having a native polypeptide sequence and structure with oneor more amino acid additions, substitutions (generally conservative innature) and/or deletions, relative to the native molecule, so long asthe modifications do not destroy immunogenic activity. The term “mutein”refers to peptides having one or more peptide mimics (“peptoids”), suchas those described in International Publication No. WO 91/04282.Preferably, the analog or mutein has at least the same immunoactivity asthe native molecule. Methods for making polypeptide analogs and muteinsare known in the art and are described further below.

As explained above, analogs generally include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

By “modified NS3” is meant an NS3 polypeptide with a modification suchthat protease activity of the NS3 polypeptide is disrupted. Themodification can include one or more amino acid additions, substitutions(generally non-conservative in nature) and/or deletions, relative to thenative molecule, wherein the protease activity of the NS3 polypeptide isdisrupted. Methods of measuring protease activity are discussed furtherbelow.

By “fragment” is intended a polypeptide consisting of only a part of theintact full-length polypeptide sequence and structure. The fragment caninclude a C-terminal deletion and/or an N-terminal deletion of thenative polypeptide. An “immunogenic fragment” of a particular HCVprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, that define an epitope, or any integerbetween 5 amino acids and the full-length sequence, provided that thefragment in question retains immunogenic activity, as measured by theassays described herein.

The term “epitope” as used herein refers to a sequence of at least about3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000amino acids (or any integer therebetween), which define a sequence thatby itself or as part of a larger sequence, binds to an antibodygenerated in response to such sequence. There is no critical upper limitto the length of the fragment, which may comprise nearly the full-lengthof the protein sequence, or even a fusion protein comprising two or moreepitopes from the HCV polyprotein. An epitope for use in the subjectinvention is not limited to a polypeptide having the exact sequence ofthe portion of the parent protein from which it is derived. Indeed,viral genomes are in a state of constant flux and contain severalvariable domains which exhibit relatively high degrees of variabilitybetween isolates. Thus the term “epitope” encompasses sequencesidentical to the native sequence, as well as modifications to the nativesequence, such as deletions, additions and substitutions (generallyconservative in nature).

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci. USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

For a description of various HCV epitopes, see, e.g., Chien et al.,Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J.Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., InternationalPublication No. WO 93/00365; Chien, D. Y., International Publication No.WO 94/01778; and U.S. Pat. Nos. 6,280,927 and 6,150,087, incorporatedherein by reference in their entireties.

As used herein, the term “conformational epitope” refers to a portion ofa full-length protein, or an analog or mutein thereof, having structuralfeatures native to the amino acid sequence encoding the epitope withinthe full-length natural protein. Native structural features include, butare not limited to, glycosylation and three dimensional structure.Preferably, a conformational epitope is produced recombinantly and isexpressed in a cell from which it is extractable under conditions whichpreserve its desired structural features, e.g. without denaturation ofthe epitope. Such cells include bacteria, yeast, insect, and mammaliancells. Expression and isolation of recombinant conformational epitopesfrom the HCV polyprotein are described in e.g., InternationalPublication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734,which applications are herein incorporated by reference in theirentirety.

As used herein the term “T-cell epitope” refers to a feature of apeptide structure which is capable of inducing T-cell immunity towardsthe peptide structure or an associated hapten. T-cell epitopes generallycomprise linear peptide determinants that assume extended conformationswithin the peptide-binding cleft of MHC molecules, (Unanue et al.,Science (1987) 236:551-557). Conversion of polypeptides to MHC classII-associated linear peptide determinants (generally between 5-14 aminoacids in length) is termed “antigen processing” which is carried out byantigen presenting cells (APCs). More particularly, a T-cell epitope isdefined by local features of a short peptide structure, such as primaryamino acid sequence properties involving charge and hydrophobicity, andcertain types of secondary structure, such as helicity, that do notdepend on the folding of the entire polypeptide. Further, it is believedthat short peptides capable of recognition by helper T-cells aregenerally amphipathic structures comprising a hydrophobic side (forinteraction with the MHC molecule) and a hydrophilic side (forinteracting with the T-cell receptor), (Margalit et al., ComputerPrediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker,Inc, ed. G. C. Woodrow et al., (1990) pp. 109-116) and further that theamphipathic structures have an α-helical configuration (see, e.g.,Spouge et al., J. Immunol. (1987) 138:204-212; Berkower et al., J.Immunol. (1986) 136:2498-2503).

Hence, segments of proteins that include T-cell epitopes can be readilypredicted using numerous computer programs. (See e.g., Margalit et al.,Computer Prediction of T-cell Epitopes, New Generation VaccinesMarcel-Dekker, Inc, ed. G. C. Woodrow et al., (1990) pp. 109-116). Suchprograms generally compare the amino acid sequence of a peptide tosequences known to induce a T-cell response, and search for patterns ofamino acids which are believed to be required for a T-cell epitope.

An “immunological response” to an HCV antigen (including bothpolypeptide and polynucleotides encoding polypeptides that are expressedin vivo) or composition is the development in a subject of a humoraland/or a cellular immune response to molecules present in thecomposition of interest. For purposes of the present invention, a“humoral immune response” refers to an immune response mediated byantibody molecules, while a “cellular immune response” is one mediatedby T-lymphocytes and/or other white blood cells. One important aspect ofcellular immunity involves an antigen-specific response by cytolyticT-cells (“CTLs”). CTLs have specificity for peptide antigens that arepresented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection oralleviation of symptoms to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

By “equivalent antigenic determinant” is meant an antigenic determinantfrom different sub-species or strains of HCV, such as from strains 1, 2,3, etc., of HCV which antigenic determinants are not necessarilyidentical due to sequence variation, but which occur in equivalentpositions in the HCV sequence in question. In general the amino acidsequences of equivalent antigenic determinants will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than30%, usually more than 40%, such as more than 60%, and even more than80-90% homology, when the two sequences are aligned.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A transcription termination sequence may belocated 3′ to the coding sequence.

A “nucleic acid” molecule or “polynucleotide” can include both double-and single-stranded sequences and refers to, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic DNA sequences fromviral (e.g. DNA viruses and retroviruses) or procaryotic DNA, andespecially synthetic DNA sequences. The term also captures sequencesthat include any of the known base analogs of DNA and RNA.

An “HCV polynucleotide” is a polynucleotide that encodes an HCVpolypeptide, as defined above.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their desiredfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper transcription factors, etc., are present. The promoter need notbe contiguous with the coding sequence, so long as it functions todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between thepromoter sequence and the coding sequence, as can transcribed introns,and the promoter sequence can still be considered “operably linked” tothe coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

A “control element” refers to a polynucleotide sequence which aids inthe expression of a coding sequence to which it is linked. The termincludes promoters, transcription termination sequences, upstreamregulatory domains, polyadenylation signals, untranslated regions,including 5′-UTRs and 3′-UTRs and when appropriate, leader sequences andenhancers, which collectively provide for the transcription andtranslation of a coding sequence in a host cell.

A “promoter” as used herein is a DNA regulatory region capable ofbinding RNA polymerase in a host cell and initiating transcription of adownstream (3′ direction) coding sequence operably linked thereto. Forpurposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eucaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

A control sequence “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. The expression cassette includes control elements,as described above, such as a promoter which is operably linked to (soas to direct transcription of) the sequence(s) or gene(s) of interest,and often includes a polyadenylation sequence as well. Within certainembodiments of the invention, the expression cassette described hereinmay be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude, one or more selectable markers, a signal which allows theplasmid construct to exist as single-stranded DNA (e.g., a M13 origin ofreplication), at least one multiple cloning site, and a “mammalian”origin of replication (e.g., a SV40 or adenovirus origin ofreplication).

“Transformation,” as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for insertion: for example, transformation by direct uptake,transfection, infection, and the like. For particular methods oftransfection, see further below. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, oralternatively, may be integrated into the host genome.

A “host cell” is a cell which has been transformed, or is capable oftransformation, by an exogenous DNA sequence.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type are present.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98%, or more, sequence identity over a defined length ofthe molecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nln.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of the antigen or antigens. The nucleic acidmolecule can be introduced directly into the recipient subject, such asby injection, inhalation, oral, intranasal and mucosal administration,or the like, or can be introduced ex vivo, into cells which have beenremoved from the host. In the latter case, the transformed cells arereintroduced into the subject where an immune response can be mountedagainst the antigen encoded by the nucleic acid molecule.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The invention described herein is intended for use in any ofthe above vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

II. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

It is a discovery of the present invention that fusion proteins,combinations of the individual components of these fusions, andpolynucleotides encoding the same, comprising an NS3, an NS4, and anNS5a polypeptide with or without a core polypeptide, or an NS3, an NS4,an NS5a, and an NS5b polypeptide, with or without a core polypeptide, ofan HCV virus can be used to activate HCV-specific T cells, i.e., T cellswhich recognize epitopes of these polypeptides.

The present invention also pertains to compositions comprising HCVnonstructural fusion proteins and HCV E1E2 complexes, as well ascompositions comprising polynucleotides encoding the same orcombinations of polypeptides and polynucleotides.

The proteins, polynucleotides, compositions and combinations of thepresent invention can be used to stimulate a cellular immune response,such as to activate HCV-specific T cells, i.e., T cells which recognizeepitopes of these polypeptides. Activation of HCV-specific T cellsprovides both in vitro and in vivo model systems for the development ofHCV vaccines, particularly for identifying HCV polypeptide epitopesassociated with a response. The compositions can also be used togenerate an immune response against HCV in a mammal, particularly a CTLresponse for either therapeutic or prophylactic purposes.

Fusion Proteins

The genomes of HCV strains contain a single open reading frame ofapproximately 9,000 to 12,000 nucleotides, which is transcribed into apolyprotein. As shown in FIG. 1 and the table below, an HCV polyprotein,upon cleavage, produces at least ten distinct products, in the order ofNH₂-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The core polypeptideoccurs at positions 1-191, numbered relative to HCV-1 (see, Choo et al.(1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, for the HCV-1 genome).This polypeptide is further processed to produce an HCV polypeptide withapproximately amino acids 1-173. The envelope polypeptides, E1 and E2,occur at about positions 192-383 and 384-746, respectively. The P7domain is found at about positions 747-809. NS2 is an integral membraneprotein with proteolytic activity and is found at about positions810-1026 of the polyprotein. NS2, in combination with NS3, (found atabout positions 1027-1657), cleaves the NS2-NS3 sissle bond which inturn generates the NS3 N-terminus and releases a large polyprotein thatincludes both serine protease and RNA helicase activities. The NS3protease, found at about positions 1027-1207, serves to process theremaining polyprotein. The helicase activity is found at about positions1193-1657. NS3 liberates an NS3 cofactor (NS4a, found about positions1658-1711), two proteins (NS4b found at about positions 1712-1972, andNS5a found at about positions 1973-2420), and an RNA-dependent RNApolymerase (NS5b found at about positions 2421-3011). Completion ofpolyprotein maturation is initiated by autocatalytic cleavage at theNS3-Ns4a junction, catalyzed by the NS3 serine protease.

Domain Approximate Boundaries* C (core)  1-191 E1 192-383 E2 384-746 P7747-809 NS2  810-1026 NS3 1027-1657 NS4a 1658-1711 NS4b 1712-1972 NS5a1973-2420 NS5b 2421-3011 *Numbered relative to HCV-1. See, Choo et al.(1991) Proc. Natl. Acad. Sci. USA 88: 2451-2455.

Fusion proteins for use in the compositions and methods, andpolynucleotides encoding therefor, include or encode an NS3 polypeptide,an NS4 (NS4a and/or NS4b) polypeptide, an NS5a polypeptide and,optionally, an NS5b polypeptide. The fusion proteins may or may notinclude all or part of the core region. In certain embodiments, none ofthe core region is present in the compositions. The nonstructuralregions need not be in the order in which they naturally occur in thenative HCV polyprotein. Thus, for example, the NS5b polypeptide may beat the N- and/or C-terminus of the fusion or may be found internally.These polypeptides may be derived from the same HCV isolate, or fromdifferent strains and isolates including isolates having any of thevarious HCV genotypes, to provide increased protection against a broadrange of HCV genotypes. Additionally, polypeptides can be selected basedon the particular viral clades endemic in specific geographic regionswhere vaccine compositions containing the fusions will be used. It isreadily apparent that the subject fusions provide an effective means oftreating HCV infection in a wide variety of contexts.

In one embodiment, the fusion protein of the present invention includesan NS3 polypeptide that has been modified to inhibit protease activity,such that further cleavage of the fusion is inhibited. The NS3polypeptide can be modified by deletion of all or a portion of the NS3protease domain. Alternatively, proteolytic activity can be inhibited bysubstitutions of amino acids within active regions of the proteasedomain. Finally, additions of amino acids to active regions of thedomain, such that the catalytic site is modified, will also serve toinhibit proteolytic activity.

As explained above, the protease activity is found at about amino acidpositions 1027-1207, numbered relative to the full-length HCV-1polyprotein (see, Choo et al., Proc. Natl. Acad. Sci. USA (1991)88:2451-2455), positions 2-182 of FIG. 3. The structure of the NS3protease and active site are known. See, e.g., De Francesco et al.,Antivir. Ther. (1998) 3:99-109; Koch et al., Biochemistry (2001)40:631-640. Thus, deletions or modifications to the native sequence willtypically occur at or near the active site of the molecule.Particularly, it is desirable to modify or make deletions to one or moreamino acids occurring at positions 1- or 2-182, preferably 1- or 2-170,or 1- or 2-155 of FIG. 3. Preferred modifications are to the catalytictriad at the active site of the protease, i.e., H, D or S residues, inorder to inactivate the protease. These residues occur at positions1083, 1105 and 1165, respectively, numbered relative to the full-lengthHCV polyprotein (positions 58, 80 and 140, respectively, of FIG. 3).Such modifications will suppress proteolytic cleavage while maintainingT-cell epitopes. One of skill in the art can readily determine portionsof the NS3 protease to delete in order to disrupt activity. The presenceor absence of activity can be determined using methods known to those ofskill in the art.

For example, protease activity or lack thereof may be determined usingassays well known in the art. See, e.g., Takeshita et al., Anal.Biochem. (1997) 247:242-246; Kakiuchi et al., J. Biochem. (1997)122:749-755; SalI et al., Biochemistry (1998) 37:3392-3401; Cho et al.,J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem.(1999) 266:192-197; Zhang et al., Anal. Biochem. (1999) 270:268-275;Kakiuchi et al., J. Virol. Meth. (1999) 80:77-84; Fowler et al., J.Biomol. Screen. (2000) 5:153-158; and Kim et al., Anal. Biochem. (2000)284:42-48.

The NS3; NS4; NS5a, and NS5b polypeptides present in the various fusionsdescribed above can either be full-length polypeptides or portions ofNS3, NS4 (NS4a and/or NS4b), NS5a, and NS5b polypeptides. The portionsof NS3, NS4, NS5a, and NS5b polypeptides making up the fusion proteinpreferably comprise at least one epitope, which is recognized by a Tcell receptor on an activated T cell, such as 2152-HEYPVGSQL-2160 (SEQID NO: 1) and/or 2224-AELIEANLLWRQEMG-2238 (SEQ ID NO:2). Epitopes ofNS3, NS4 (NS4a and NS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b canbe identified by several methods. For example, NS3, NS4, NS5a, NS5bpolypeptides or fusion proteins comprising any combination of the above,can be isolated, for example, by immunoaffinity purification using amonoclonal antibody for the polypeptide or protein. The isolated proteinsequence can then be screened by preparing a series of short peptides byproteolytic cleavage of the purified protein, which together span theentire protein sequence. By starting with, for example, 100-merpolypeptides, each polypeptide can be tested for the presence ofepitopes recognized by a T-cell receptor on an HCV-activated T cell,progressively smaller and overlapping fragments can then be tested froman identified 100-mer to map the epitope of interest.

Epitopes recognized by a T-cell receptor on an HCV-activated T cell canbe identified by, for example, ⁵¹Cr release assay or bylymphoproliferation assay (see the examples). In a ⁵¹Cr release assay,target cells can be constructed that display the epitope of interest bycloning a polynucleotide encoding the epitope into an expression vectorand transforming the expression vector into the target cells.HCV-specific CD8⁺ T cells will lyse target cells displaying, forexample, an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitopeand will not lyse cells that do not display such an epitope. In alymphoproliferation assay, HCV-activated CD4⁺ T cells will proliferatewhen cultured with, for example, an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, orNS3NS4NS5aNS5b epitopic peptide, but not in the absence of an HCVepitopic peptide.

NS3, NS4, NS5a, and NS5b polypeptides can occur in any order in thefusion protein. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore of one or more of the polypeptides may occur in the fusion protein.Multiple viral strains of HCV occur, and NS3, NS4, NS5a, and NS5bpolypeptides of any of these strains can be used in a fusion protein. Arepresentative fusion protein for use in the present invention is shownif FIGS. 5A-5J. The depicted sequence includes amino acids 1242-3011 ofthe HCV polyprotein (representing polypeptides from NS3, NS4, NS5a andNS5b) with amino acids 1-121 of the HCV polyprotein (representing apolypeptide from the core region) fused to the C-terminus of NS5b. Thisnumbering is relative to the HCV-1 polyprotein.

Nucleic acid and amino acid sequences of a number of HCV strains andisolates, including nucleic acid and amino acid sequences of NS3, NS4,NS5a, NS5b genes and polypeptides have been determined. For example,isolate HCV J1.1 is described in Kubo et al. (1989) Japan. Nucl. AcidsRes. 17:10367-10372; Takeuchi et al. (1990) Gene 91:287-291; Takeuchi etal. (1990) J. Gen. Virol. 71:3027-3033; and Takeuchi et al. (1990) Nucl.Acids Res. 18:4626. The complete coding sequences of two independentisolates, HCV-J and BK, are described by Kato et al., (1990) Proc. Natl.Acad. Sci. USA 87:9524-9528 and Takamizawa et al., (1991) J. Virol.65:1105-1113 respectively.

Publications that describe HCV-1 isolates include Choo et al. (1990)Brit. Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad. Sci.USA 88:2451-2455 and Han et al. (1991) Proc. Natl. Acad. Sci. USA88:1711-1715. HCV isolates HC-J1 and HC-J4 are described in Okamoto etal. (1991) Japan J. Exp. Med. 60:167-177. HCV isolates HCT 18˜, HCT 23,Th, HCT 27, EC1 and EC10 are described in Weiner et al. (1991) Virol.180:842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 are described inEnomoto et al. (1990) Biochem. Biophys. Res. Commun. 170:1021-1025. HCVisolates A, C, D & E are described in Tsukiyama-Kohara et al. (1991)Virus Genes 5:243-254.

Each of the NS3, NS4, NS5a, and NS5b components of a fusion protein canbe obtained from the same HCV strain or isolate or from different HCVstrains or isolates. Fusion proteins comprising HCV polypeptides from,for example, the NS3 polypeptide can be derived from a first strain ofHCV, and the NS4, and NS5a polypeptides can be derived from a secondstrain of HCV. Alternatively, the NS4 polypeptide can be derived from afirst strain of HCV, and the NS3 and NS5a polypeptides can be derivedfrom a second strain of HCV. Optionally, the NS5a polypeptide can bederived from a first strain of HCV, and the NS3 and NS4 polypeptides canbe derived from a second strain of HCV. NS3, NS4 and NS5a polypeptidesthat are each derived from different HCV strains can also be used in anHCV fusion protein. Similarly, in a fusion protein comprising NS5b, atleast one of the NS3, NS4, NS5a, and NS5b polypeptides can be derivedfrom a different HCV strain than the other polypeptides. Optionally,NS3, NS4, NS5a, and NS5b polypeptides that are each derived fromdifferent HCV strains can also be used in an NS3NS4NS5aNS5b fusionprotein.

In addition to NS3, NS4a, NS4b, NS5a and NS5b, the fusion proteins cancontain other polypeptides derived from the HCV polyprotein. Forexample, it may be desirable to include polypeptides derived from thecore region of the HCV polyprotein. This region occurs at amino acidpositions 1-191 of the HCV polyprotein, numbered relative to HCV-1.Either the full-length protein, fragments thereof, such as amino acids1-150, e.g., amino acids 1-130, 1-120, for example, amino acids 1-121,1-122, 1-123, etc., or smaller fragments containing epitopes of thefull-length protein may be used in the subject fusions, such as thoseepitopes found between amino acids 10-53, amino acids 10-45, amino acids67-88, amino acids 120-130, or any of the core epitopes identified in,e.g., Houghton et al., U.S. Pat. No. 5,350,671; Chien et al., Proc.Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent.Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO93/00365; Chien, D. Y., International Publication No. WO 94/01778; andU.S. Pat. Nos. 6,280,927 and 6,150,087, the disclosures of which areincorporated herein by reference in their entireties. Moreover, aprotein resulting from a frameshift in the core region of thepolyprotein, such as described in International Publication No. WO99/63941, may be used. The fusions may also contain polynucleotidesencoding E1E2 polypeptides, as described further below.

Preferably, the above-described fusion proteins, as well as theindividual components of these proteins, are produced recombinantly. Apolynucleotide encoding these proteins can be introduced into anexpression vector which can be expressed in a suitable expressionsystem. A variety of bacterial, yeast, mammalian and insect expressionsystems are available in the art and any such expression system can beused. Optionally, a polynucleotide encoding these proteins can betranslated in a cell-free translation system. Such methods are wellknown in the art. The proteins also can be constructed by solid phaseprotein synthesis.

If desired, the fusion proteins, or the individual components of theseproteins, also can contain other non-HCV amino acid sequences, such asamino acid linkers or signal sequences, as well as ligands useful inprotein purification, such as glutathione-S-transferase andstaphylococcal protein A.

E1E2 Polypeptides

As explained above, the compositions of the present invention may alsoinclude E1 and E2 polypeptides, complexes of these polypeptides orpolynucleotides encoding the same. The E1 and E2 polypeptides andcomplexes thereof can be provided independent of the nonstructuralfusion protein or can be incorporated into the same fusion. Moreover,E1E2 complexes can be provided as proteins, or as polynucleotidesencoding the same.

In this regard, E1, E2 and p7 are known to contain human T-cell epitopes(both CD4+ and CD8+) and including one or more of these epitopes servesto increase vaccine efficacy as well as to increase protective levelsagainst multiple HCV genotypes. Moreover, multiple copies of specific,conserved T-cell epitopes can also be used in E1E2 complexes, such as acomposite of epitopes from different genotypes.

As explained above, the E1 and E2 polypeptides that make up the E1E2complexes can be associated either through non-covalent or covalentinteractions. Such complexes may be made up of immunogenic fragments ofE1 and E2 which comprise epitopes. For example, fragments of E1polypeptides can comprise from about 5 to nearly the full-length of themolecule, such as 6, 10, 25, 50, 75, 100, 125, 150, 175, 185 or moreamino acids of an E1 polypeptide, or any integer between the statednumbers. Similarly, fragments of E2 polypeptides can comprise 6, 10, 25,50, 75, 100, 150, 200, 250, 300, or 350 amino acids of an E2polypeptide, or any integer between the stated numbers. The E1 and E2polypeptides may be from the same or different HCV strains. For example,epitopes derived from, e.g., the hypervariable region of E2, such as aregion spanning amino acids 384-410 or 390-410, can be included in theE2 polypeptide. A particularly effective E2 epitope to incorporate intothe E2 sequence or E1E2 complexes is one which includes a consensussequence derived from this region, such as the consensus sequenceGly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn(SEQ ID NO:5), which represents a consensus sequence for amino acids390-410 of the HCV type 1 genome. Additional epitopes of E1 and E2 areknown and described in, e.g., Chien et al., International PublicationNo. WO 93/00365, incorporated by reference herein in its entirety.

Moreover, the E1 and E2 polypeptides may lack all or a portion of themembrane spanning domain. The membrane anchor sequence functions toassociate the polypeptide to the endoplasmic reticulum. Normally, suchpolypeptides are capable of secretion into growth medium in which anorganism expressing the protein is cultured. However, as described inInternational Publication No. WO 98/50556, such polypeptides may also berecovered intracellularly. Secretion into growth medium is readilydetermined using a number of detection techniques, including, e.g.,polyacrylamide gel electrophoresis and the like, and immunologicaltechniques such as immunoprecipitation assays as described in, e.g.,International Publication No. WO 96/04301, published Feb. 15, 1996. WithE1, generally polypeptides terminating with about amino acid position370 and higher (based on the numbering of HCV1 E1) will be retained bythe ER and hence not secreted into growth media. With E2, polypeptidesterminating with about amino acid position 731 and higher (also based onthe numbering of the HCV1 E2 sequence) will be retained by the ER andnot secreted. (See, e.g., International Publication No. WO 96/04301,published Feb. 15, 1996). It should be noted that these amino acidpositions are not absolute and may vary to some degree. Thus, thepresent invention contemplates the use of E1 and E2 polypeptides whichretain the transmembrane binding domain, as well as polypeptides whichlack all or a portion of the transmembrane binding domain, including E1polypeptides terminating at about amino acids 369 and lower, and E2polypeptides, terminating at about amino acids 730 and lower, areintended to be captured by the present invention. Furthermore, theC-terminal truncation can extend beyond the transmembrane spanningdomain towards the N-terminus. Thus, for example, E1 truncationsoccurring at positions lower than, e.g., 360 and E2 truncationsoccurring at positions lower than, e.g., 715, are also encompassed bythe present invention. All that is necessary is that the truncated E1and E2 polypeptides remain functional for their intended purpose.However, particularly preferred truncated E1 constructs are those thatdo not extend beyond about amino acid 300. Most preferred are thoseterminating at position 360. Preferred truncated E2 constructs are thosewith C-terminal truncations that do not extend beyond about amino acidposition 715. Particularly preferred E2 truncations are those moleculestruncated after any of amino acids 715-730, such as 725. If truncatedmolecules are used, it is preferable to use E1 and E2 molecules that areboth truncated.

E2 exists as multiple species (Spaete et al., Virol. (1992) 188:819-830;Selby et al., J. Virol. (1996) 70:5177-5182; Grakoui et al., J. Virol.(1993) 67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026) andclipping and proteolysis may occur at the N- and C-termini of the E1 andE2 polypeptides. Thus, an E2 polypeptide for use herein may comprise atleast amino acids 405-661, e.g., 400,401, 402 . . . to 661, such as384-661, 384-715, 384-746, 384-749 or 384-809, or 384 to any C-terminusbetween 661-809, of an HCV polyprotein, numbered relative to thefull-length HCV-1 polyprotein. Similarly, preferable E1 polypeptides foruse herein can comprise amino acids 192-326, 192-330, 192-333, 192-360,192-363, 192-383, or 192 to any C-terminus between 326-383, of an HCVpolyprotein.

The E1 and E2 polypeptides and complexes thereof may also be present asasialoglycoproteins. Such asialoglycoproteins are produced by methodsknown in the art, such as by using cells in which terminal glycosylationis blocked. When these proteins are expressed in such cells and isolatedby GNA lectin affinity chromatography, the E1 and E2 proteins aggregatespontaneously. Detailed methods for producing these E1E2 aggregates aredescribed in, e.g., U.S. Pat. No. 6,074,852, incorporated herein byreference in its entirety. For example, E1E2 complexes are readilyproduced recombinantly, either as fusion proteins or by e.g.,co-transfecting host cells with constructs encoding for the E1 and E2polypeptides of interest. Co-transfection can be accomplished either intrans or cis, i.e., by using separate vectors or by using a singlevector which bears both of the E1 and E2 genes. If done using a singlevector, both genes can be driven by a single set of control elements or,alternatively, the genes can be present on the vector in individualexpression cassettes, driven by individual control elements. Followingexpression, the E1 and E2 proteins will spontaneously associate.Alternatively, the complexes can be formed by mixing the individualproteins together which have been produced separately, either inpurified or semi-purified form, or even by mixing culture media in whichhost cells expressing the proteins, have been cultured, if the proteinsare secreted. Finally, the E1E2 complexes of the present invention maybe expressed as a fusion protein wherein the desired portion of E1 isfused to the desired portion of E2.

Moreover, the E1E2 complexes may be present as a heterogeneous mixtureof molecules, due to clipping and proteolytic cleavage, as describedabove. Thus, a composition including E1E2 complexes may include multiplespecies of E1E2, such as E1E2 terminating at amino acid 746 (E1E2₇₄₆),E1E2 terminating at amino acid 809 (E1E2₈₀₉), or any of the othervarious E1 and E2 molecules described above, such as E2 molecules withN-terminal truncations of from 1-20 amino acids, such as E2 speciesbeginning at amino acid 387, amino acid 402, amino acid 403, etc.

E1E2 complexes are readily produced recombinantly, either as fusionproteins or by e.g., co-transfecting host cells with constructs encodingfor the E1 and E2 polypeptides of interest. Co-transfection can beaccomplished either in trans or cis, i.e., by using separate vectors orby using a single vector which bears both of the E1 and E2 genes. Ifdone using a single vector, both genes can be driven by a single set ofcontrol elements or, alternatively, the genes can be present on thevector in individual expression cassettes, driven by individual controlelements. Following expression, the E1 and E2 proteins willspontaneously associate. Alternatively, the complexes can be formed bymixing the individual proteins together which have been producedseparately, either in purified or semi-purified form, or even by mixingculture media in which host cells expressing the proteins, have beencultured, if the proteins are secreted. Finally, the E1E2 complexes ofthe present invention may be expressed as a fusion protein wherein thedesired portion of E1 is fused to the desired portion of E2.

Methods for producing E1E2 complexes from full-length, truncated E1 andE2 proteins which are secreted into media, as well as intracellularlyproduced truncated proteins, are known in the art. For example, suchcomplexes may be produced recombinantly, as described in U.S. Pat. No.6,121,020; Ralston et al., J. Virol. (1993) 67:6753-6761, Grakoui etal., J Virol. (1993) 67:1385-1395; and Lanford et al., Virology (1993)197:225-235.

Polynucleotides Encoding the Fusion Proteins and E1E2 Complexes

Polynucleotides contain less than an entire HCV genome and can be RNA orsingle- or double-stranded DNA. Preferably, the polynucleotides areisolated free of other components, such as proteins and lipids. Thepolynucleotides encode the fusion proteins, E1 and E2 polypeptides andcomplexes thereof, described above, and thus comprise coding sequencesthereof. Polynucleotides of the invention can also comprise othernon-HCV nucleotide sequences, such as sequences coding for linkers,signal sequences, or ligands useful in protein purification such asglutathione-S-transferase and staphylococcal protein A.

Polynucleotides encoding the various HCV polypeptides can be isolatedfrom a genomic library derived from nucleic acid sequences present in,for example, the plasma, serum, or liver homogenate of an HCV infectedindividual or can be synthesized in the laboratory, for example, usingan automatic synthesizer. An amplification method such as PCR can beused to amplify polynucleotides from either HCV genomic DNA or cDNAencoding therefor.

Polynucleotides can comprise coding sequences for these polypeptideswhich occur naturally or can include artificial sequences which do notoccur in nature. These polynucleotides can be ligated to form a codingsequence for the fusion proteins and E1E2 complexes using standardmolecular biology techniques. If desired, polynucleotides can be clonedinto an expression vector and transformed into, for example, bacterial,yeast, insect, or mammalian cells so that the fusion proteins of theinvention can be expressed in and isolated from a cell culture.

The expression constructs of the present invention, including thedesired fusion, or individual expression constructs comprising theindividual components of these fusions, may be used for nucleic acidimmunization, to stimulate an immunological response, such as a cellularimmune response, using standard gene delivery protocols. Methods forgene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466, incorporated by reference herein in theirentireties. Genes can be delivered either directly to the vertebratesubject or, alternatively, delivered ex vivo, to cells derived from thesubject and the cells reimplanted in the subject. For example, theconstructs can be delivered as plasmid DNA, e.g., contained within aplasmid, such as pBR322, pUC, or ColE1

Additionally, the expression constructs can be packaged in liposomesprior to delivery to the cells. Lipid encapsulation is generallyaccomplished using liposomes which are able to stably bind or entrap andretain nucleic acid. The ratio of condensed DNA to lipid preparation canvary but will generally be around 1:1 (mg DNA:micromoles lipid), or moreof lipid. For a review of the use of liposomes as carriers for deliveryof nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp. 512-527.

Liposomal preparations for use with the present invention includecationic (positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can beprepared from readily available materials using techniques well known inthe art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978)75:4194-4198; PCT Publication No. WO 90/11092 for a description of thesynthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes. The various liposome-nucleic acid complexes are preparedusing methods known in the art. See, e.g., Straubinger et al., inMETHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc.Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al.,Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979)17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostroet al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al.,Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter,Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem.(1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA(1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

The DNA can also be delivered in cochleate lipid compositions similar tothose described by Papahadjopoulos et al., Biochem. Biophys. Acta.(1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems, such as murine sarcoma virus, mousemammary tumor virus, Moloney murine leukemia virus, and Rous sarcomavirus. A selected gene can be inserted into a vector and packaged inretroviral particles using techniques known in the art. The recombinantvirus can then be isolated and delivered to cells of the subject eitherin vivo or ex vivo. A number of retroviral systems have been described(U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989)7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa etal., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci.USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet.Develop. (1993) 3:102-109. Briefly, retroviral gene delivery vehicles ofthe present invention may be readily constructed from a wide variety ofretroviruses, including for example, B, C, and D type retroviruses aswell as spumaviruses and lentiviruses such as FIV, HIV, HIV-1, HIV-2 andSIV (see RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolatedfrom known sources using commonly available techniques.

A number of adenovirus vectors have also been described, such asadenovirus Type 2 and Type 5 vectors. Unlike retroviruses whichintegrate into the host genome, adenoviruses persist extrachromosomallythus minimizing the risks associated with insertional mutagenesis(Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J.Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994)5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., GeneTherapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629;and Rich et al., Human Gene Therapy (1993) 4:461-476).

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as but not limited to vectorsderived from the Sindbis and Semliki Forest viruses, VEE, will also finduse as viral vectors for delivering the gene of interest. For adescription of Sindbis-virus derived vectors useful for the practice ofthe instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519;and International Publication Nos. WO 95/07995 and WO 96/17072.

Other vectors can be used, including but not limited to simian virus 40and cytomegalovirus. Bacterial vectors, such as Salmonella ssp. Yersiniaenterocolitica, Shigella spp., Vibrio cholerae, Mycobacterium strainBCG, and Listeria monocytogenes can be used. Minichromosomes such as MCand MC1, bacteriophages, cosmids (plasmids into which phage lambda cossites have been inserted) and replicons (genetic elements that arecapable of replication under their own control in a cell) can also beused.

The expression constructs may also be encapsulated, adsorbed to, orassociated with, particulate carriers. Such carriers present multiplecopies of a selected molecule to the immune system and promote trappingand retention of molecules in local lymph nodes. The particles can bephagocytosed by macrophages and can enhance antigen presentation throughcytokine release. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al.,J. Microencap. (1996).

One preferred method for adsorbing macromolecules onto preparedmicroparticles is described in International Publication No. WO00/050006, incorporated herein by reference in its entirety. Briefly,microparticles are rehydrated and dispersed to an essentially monomericsuspension of microparticles using dialyzable anionic or cationicdetergents. Useful detergents include, but are not limited to, any ofthe various N-methylglucamides (known as MEGAs), such asheptanoyl-N-methylglucamide (MEGA-7), octanoyl-N-methylglucamide(MEGA-8), nonanoyl-N-methylglucamide (MEGA-9), anddecanoyl-N-methyl-glucamide (MEGA-10); cholic acid; sodium cholate;deoxycholic acid; sodium deoxycholate; taurocholic acid; sodiumtaurocholate; taurodeoxycholic acid; sodium taurodeoxycholate;3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS);3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate(CHAPSO); -dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate(ZWITTERGENT 3-12); N,N-bis-(3-D-gluconeamidopropyl)-deoxycholamide(DEOXY-BIGCHAP); -octylglucoside; sucrose monolaurate; glycocholicacid/sodium glycocholate; laurosarcosine (sodium salt); glycodeoxycholicacid/sodium glycodeoxycholate; sodium dodceyl sulfate (SDS);3-(trimethylsilyl)-1-propanesulfonic acid (DSS); cetrimide (CTAB, theprincipal component of which is hexadecyltrimethylammonium bromide);hexadecyltrimethylammonium bromide; dodecyltrimethylammonium bromide;hexadecyltrimethyl-ammonium bromide; tetradecyltrimethylammoniumbromide; benzyl dimethyldodecylammonium bromide; benzyldimethyl-hexadecylammonium chloride; and benzyldimethyltetra-decylammonium bromide. The above detergents arecommercially available from e.g., Sigma Chemical Co., St. Louis, Mo.Various cationic lipids known in the art can also be used as detergents.See Balasubramaniam et al., 1996, Gene Ther., 3:163-72 and Gao, X., andL. Huang. 1995, Gene Ther., 2:7110-722.

A wide variety of other methods can be used to deliver the expressionconstructs to cells. Such methods include DEAE dextran-mediatedtransfection, calcium phosphate precipitation, polylysine- orpolyornithine-mediated transfection, or precipitation using otherinsoluble inorganic salts, such as strontium phosphate, aluminumsilicates including bentonite and kaolin, chromic oxide, magnesiumsilicate, talc, and the like. Other useful methods of transfectioninclude electroporation, sonoporation, protoplast fusion, liposomes,peptoid delivery, or microinjection. See, e.g., Sambrook et al., supra,for a discussion of techniques for transforming cells of interest; andFelgner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for areview of delivery systems useful for gene transfer. Methods ofdelivering DNA using electroporation are described in, e.g., U.S. Pat.Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent PublicationNo. 2002/0146831; and International Publication No. WO/0045823, all ofwhich are incorporated herein by reference in their entireties.

Moreover, the HCV polynucleotides can be adsorbed to, or entrappedwithin, an ISCOM. Classic ISCOMs are formed by combination ofcholesterol, saponin, phospholipid, and immunogens, such as viralenvelope proteins. Generally, the HCV molecules (usually with ahydrophobic region) are solubilized in detergent and added to thereaction mixture, whereby ISCOMs are formed with the HCV moleculeincorporated therein. ISCOM matrix compositions are formed identically,but without viral proteins. Proteins with high positive charge may beelectrostatically bound in the ISCOM particles, rather than throughhydrophobic forces. For a more detailed general discussion of saponinsand ISCOMs, and methods of formulating ISCOMs, see Barr et al. (1998)Adv. Drug Delivery Reviews 32:247-271 (1998); U.S. Pat. Nos. 4,981,684,5,178,860, 5,679,354 and 6,027,732, incorporated herein by reference intheir entireties; European Publ. Nos. EPA 109,942; 180,564 and 231,039;and Coulter et al. (1998) Vaccine 16:1243.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for delivering theexpression constructs of the present invention. The particles are coatedwith the construct to be delivered and accelerated to high velocity,generally under a reduced atmosphere, using a gun powder discharge froma “gene gun.” For a description of such techniques, and apparatusesuseful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;5,100,792; 5,179,022; 5,371,015; and 5,478,744.

Compositions Comprising Fusion Proteins or Polynucleotides

The invention also provides compositions comprising the fusion proteinsor polynucleotides, as well as compositions including the individualcomponents of these fusion proteins or polynucleotides. Compositions ofthe invention preferably comprise a pharmaceutically acceptable carrier.The carrier should not itself induce the production of antibodiesharmful to the host. Pharmaceutically acceptable carriers are well knownto those in the art. Such carriers include, but are not limited to,large, slowly metabolized, macromolecules, such as proteins,polysaccharides such as latex functionalized sepharose, agarose,cellulose, cellulose beads and the like, polylactic acids, polyglycolicacids, polymeric amino acids such as polyglutamic acid, polylysine, andthe like, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, glycerol, dextrose, ethanol, or the like, singlyor in combination, as well as substances such as wetting agents,emulsifying agents, or pH buffering agents. Liposomes can also be usedas a carrier for a composition of the invention, such liposomes aredescribed above.

If desired, co-stimulatory molecules which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asGM-CSF, IL-2, and IL-12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants which can be used include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (U.S. Pat. No. 6,299,884, incorporated herein by reference in itsentirety; Chapter 10 in Vaccine design: the subunit and adjuvantapproach, eds. Powell & Newman, Plenum Press 1995), containing 5%Squalene, 0.5% TWEEN 80™, and 0.5% SPAN 85™ (optionally containingvarious amounts of MTP-PE (see below), although not required) formulatedinto submicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% TWEEN 80™, 5% pluronic-blocked polymer L121, and thr-MDPeither microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN80™, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (3) saponinadjuvants, such as QS21 or STIMULON™ (Cambridge Bioscience, Worcester,Mass.) may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes), which ISCOMs may be devoid of additionaldetergent, see, e.g., International Publication No. WO 00/07621; (4)Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA);(5) cytokines, such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7,IL-12 (International Publication No. WO 99/44636), etc.), interferons(e.g., gamma interferon), macrophage colony stimulating factor (M-CSF),tumor necrosis factor (TNF), etc.; (6) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (wherelysine is substituted for the wild-type amino acid at position 63)LT-R72 (where arginine is substituted for the wild-type amino acid atposition 72), CT-S109 (where serine is substituted for the wild-typeamino acid at position 109), and PT-K9/G129 (where lysine is substitutedfor the wild-type amino acid at position 9 and glycine substituted atposition 129) (see, e.g., International Publication Nos. W093/13202 andW092/19265); (7) MPL or 3-O-deacylated MPL (3dMPL) (see, e.g., GB2220221), EP-A-0689454, optionally in the substantial absence of alumwhen used with pneumococcal saccharides (see, e.g., InternationalPublication No. WO 00/56358); (8) combinations of 3dMPL with, forexample, QS21 and/or oil-in-water emulsions (see, e.g., EP-A-0835318,EP-A-0735898, EP-A-0761231; (9) oligonucleotides comprising CpG motifs(see, e.g., Roman et al. (1997) Nat. Med. 3:849-854; Weiner et al.(1997) Proc. Natl. Acad. Sci. USA 94:10833-10837; Davis et al. (1998) J.Immunol. 160:870-876; Chu et al. (1997) J. Exp. Med. 186:1623-1631;Lipford et al. (1997) Eur. J. Immunol. 27:2340-2344; Moldoveanu et al.(1988) Vaccine 16:1216-1224; Krieg et al. (1995) Nature 374:546-549;Klinman et al. (1996) Proc. Natl. Acad. Sci. USA 93:2879-2883; Ballas etal. (1996) J. Immunol. 157:1840-1845; Cowdery et al. (1996) J. Immunol.156:4570-4575; Halpern et al. (1996) Cell Immunol. 167:72-78; Yamamotoet al. (1988) Jpn. J. Cancer Res. 79:866-873; Stacey et al. (1996) J.Immunol. 157:2116-2122; Messina et al. (1991) J. Immunol. 147:1759-1764;Yi et al. (1996) J. Immunol. 157:4918-4925; Yi et al. (1996) J. Immunol.157:5394-5402; Yi et al. (1998) J. Immunol. 160:4755-4761; Yi et al.(1998) J. Immunol. 160:5898-5906; International Publication Nos. WO96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO98/37919 and WO 98/52581), such as those containing at least on CGdinucleotide, with cytosine optionally replaced with 5-methylcytosine;(10) a polyoxyethylene ether or a polyoxyethylene ester (see, e.g.,International Publication No. WO 99/52549); (11) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (see, e.g.,International Publication No. WO 01/21207) or a polyoxyethylene alkylether or ester surfactant in combination with at least one additionalnon-ionic surfactant such as an octoxynol (see, e.g., InternationalPublication No. WO 01/21152); (12) a saponin and an immunostimulatoryoligonucleotide such as a CpG oligonucleotide (see, e.g., InternationalPublication No. WO 00/62800); (13) an immunostimulant and a particle ofmetal salt (see, e.g., International Publication No. WO 00/23105); and(14) other substances that act as immunostimulating agents to enhancethe effectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Moreover, the HCV proteins can be adsorbed to, or entrapped within, anISCOM, as described above. Additionally, ISCOMs with adsorbed HCV coreproteins, either the entire core region or a fragment of HCV coreprotein, may be added to the formulations. Most preferably, the HCV coreprotein is a fragment comprising a polypeptide from the region spanningamino acid positions 121-135. See, e.g., International Publication No.WO 01/37869A, incorporated herein by reference in its entirety.

As explained above, the composition may also contain immunostimulatorymolecules, either in addition to or in place of the antigen deliverysystem. Immunostimulatory agents for use herein include, withoutlimitation, monophosphorylipid A (MPL), trehalose dimycolate (TDM), andcell wall skeleton (CWS), preferably MPL+CWS (Detox™). MPL may beformulated into an emulsion to enhance its immunostimulatory affect.See, e.g., Ulrich et al., “MPLr immunostimulat: adjuvant formulations.”in Vaccine Adjuvants: Prepartion Methods and Research Protocols (O'HaganD T, ed.) Human Press Inc., NJ (2000) pp. 273-282. MPL has been shown toinduce the synthesis and release of cytokines, particularly IL-2 andIFN-γ. Other useful immunostimulatory molecules include LPS andimmunostimulatory nucleic acid sequences (ISS), including but notlimited to, unmethylated CpG motifs, such as CpG oligonucleotides.

Oligonucleotides containing unmethylated CpG motifs have been shown toinduce activation of B cells, NK cells and antigen-presenting cells(APCs), such as monocytes and macrophages. See, e.g., U.S. Pat. No.6,207,646. Thus, adjuvants derived from the CpG family of molecules, CpGdinucleotides and synthetic oligonucleotides which comprise CpG motifs(see, e.g., Krieg et al. Nature (1995) 374:546 and Davis et al. J.Immunol. (1998) 160:870-876) such as any of the variousimmunostimulatory CpG oligonucleotides disclosed in U.S. Pat. No.6,207,646, may be used in the subject methods and compositions. Such CpGoligonucleotides generally comprise at least 8 up to about 100basepairs, preferably 8 to 40 basepairs, more preferably 15-35basepairs, preferably 15-25 basepairs, and any number of basepairsbetween these values. For example, oligonucleotides comprising theconsensus CpG motif, represented by the formula 5′-X₁CGX₂-3′, where X₁and X₂ are nucleotides and C is unmethylated, will find use asimmunostimulatory CpG molecules. Generally, X₁ is A, G or T, and X₂ is Cor T. Other useful CpG molecules include those captured by the formula5′-X₁X₂CGX₃X₄, where X₁ and X₂ are a sequence such as GpT, GpG, GpA,ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT or TpG, and X₃ and X₄ are TpT,CpT, ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, or TpG, wherein“p” signifies a phosphate bond. Preferably, the oligonucleotides do notinclude a GCG sequence at or near the 5′- and/or 3′ terminus.Additionally, the CpG is preferably flanked on its 5′-end with twopurines (preferably a GpA dinucleotide) or with a purine and apyrimidine (preferably, GpT), and flanked on its 3′-end with twopyrimidines, preferably a TpT or TpC dinucleotide. Thus, preferredmolecules will comprise the sequence GACGTT, GACGTC, GTCGTT or GTCGCT,and these sequences will be flanked by several additional nucleotides.The nucleotides outside of this central core area appear to be extremelyamendable to change.

Moreover, the CpG oligonucleotides for use herein may be double- orsingle-stranded. Double-stranded molecules are more stable in vivo whilesingle-stranded molecules display enhanced immune activity.Additionally, the phosphate backbone may be modified, such asphosphorodithioate-modified, in order to enhance the immunostimulatoryactivity of the CpG molecule. As described in U.S. Pat. No. 6,207,646,CpG molecules with phosphorothioate backbones preferentially activateB-cells, while those having phosphodiester backbones preferentiallyactivate monocytic (macrophages, dendritic cells and monocytes) and NKcells.

One exemplary CpG oligonucleotide for use in the present compositionshas the sequence 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO:6).

CpG molecules can readily be tested for their ability to stimulate animmune response using standard techniques, well known in the art. Forexample, the ability of the molecule to stimulate a humoral and/orcellular immune response is readily determined using the immunoassaysdescribed above. Moreover, the antigen and adjuvant compositions can beadministered with and without the CpG molecule to determine whether animmune response is enhanced.

The HCV proteins may also be encapsulated, adsorbed to, or associatedwith, particulate carriers, as described above with reference to the HCVpolynucleotides. As explained above, examples of particulate carriersinclude those derived from polymethyl methacrylate polymers, as well asmicroparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,Pharm. Res. (1993) 10:362-368; and McGee et al., J. Microencap. (1996).One preferred method for adsorbing macromolecules onto preparedmicroparticles is described above and in International Publication No.WO 00/050006, incorporated herein by reference in its entirety.

Methods of Producing HCV-Specific Antibodies

The HCV proteins can be used to produce HCV-specific polyclonal andmonoclonal antibodies. HCV-specific polyclonal and monoclonal antibodiesspecifically bind to HCV antigens. Polyclonal antibodies can be producedby administering the fusion protein to a mammal, such as a mouse, arabbit, a goat, or a horse. Serum from the immunized animal is collectedand the antibodies are purified from the plasma by, for example,precipitation with ammonium sulfate, followed by chromatography,preferably affinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art.

Monoclonal antibodies directed against HCV-specific epitopes present inthe proteins can also be readily produced. Normal B cells from a mammal,such as a mouse, immunized with an HCV protein, can be fused with, forexample, HAT-sensitive mouse myeloma cells to produce hybridomas.Hybridomas producing HCV-specific antibodies can be identified using RIAor ELISA and isolated by cloning in semi-solid agar or by limitingdilution. Clones producing HCV-specific antibodies are isolated byanother round of screening.

Antibodies, either monoclonal and polyclonal, which are directed againstHCV epitopes, are particularly useful for detecting the presence of HCVor HCV antigens in a sample, such as a serum sample from an HCV-infectedhuman. An immunoassay for an HCV antigen may utilize one antibody orseveral antibodies. An immunoassay for an HCV antigen may use, forexample, a monoclonal antibody directed towards an HCV epitope, acombination of monoclonal antibodies directed towards epitopes of oneHCV polypeptide, monoclonal antibodies directed towards epitopes ofdifferent HCV polypeptides, polyclonal antibodies directed towards thesame HCV antigen, polyclonal antibodies directed towards different HCVantigens, or a combination of monoclonal and polyclonal antibodies.Immunoassay protocols may be based, for example, upon competition,direct reaction, or sandwich type assays using, for example, labeledantibody. The labels may be, for example, fluorescent, chemiluminescent,or radioactive.

The polyclonal or monoclonal antibodies may further be used to isolateHCV particles or antigens by immunoaffinity columns. The antibodies canbe affixed to a solid support by, for example, adsorption or by covalentlinkage so that the antibodies retain their immunoselective activity.Optionally, spacer groups may be included so that the antigen bindingsite of the antibody remains accessible. The immobilized antibodies canthen be used to bind HCV particles or antigens from a biological sample,such as blood or plasma. The bound HCV particles or antigens arerecovered from the column matrix by, for example, a change in pH.

HCV-Specific T Cells

HCV-specific T cells that are activated by the above-described fusionsand E1E2 complexes, including the NS3NS4NS5a fusion protein orNS3NS4NS5aNS5b fusion protein, and the E1E2 complexes, expressed in vivoor in vitro, preferably recognize an epitope of an HCV polypeptide suchas an E1, E2, NS3, NS4, NS5a, NS5b polypeptide, including an epitope ofan NS3NS4NS5a fusion protein or an NS3NS4NS5aNS5b fusion protein, or anE1E2 complex. HCV-specific T cells can be CD8⁺ or CD4⁺.

HCV-specific CD8⁺ T cells preferably are cytotoxic T lymphocytes (CTL)which can kill HCV-infected cells that display E1, E2, NS3, NS4, NS5a,NS5b epitopes complexed with an MHC class I molecule. HCV-specific CD8⁺T cells may also express interferon-γ (IFN-γ). HCV-specific CD8⁺ T cellscan be detected by, for example, ⁵¹Cr release assays (see the examples).⁵¹Cr release assays measure the ability of HCV-specific CD8⁺ T cells tolyse target cells displaying an E1, E2, E1E2, NS3, NS4, NS5a, NS5b,NS3NS4NS5a, or NS3NS4NS5aNS5b epitope. HCV-specific CD8⁺ T cells whichexpress IFN-γ can also be detected by immunological methods, preferablyby intracellular staining for IFN-γ after in vitro stimulation with anE1, E2, NS3, an NS4, an NS5a, or an NS5b polypeptide (see the examples).

HCV-specific CD4⁺ cells activated by the above-described E1E2 complexesand fusions, such as an E1 polypeptide, an E2 polypeptide, an E1E2complex, NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, expressed in vivoor in vitro, preferably recognize an epitope of an E1, E2, NS3, NS4,NS5a, or NS5b polypeptide, including an epitope of an E1E2 complex,NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, that is bound to an MHCclass II molecule on an HCV-infected cell and proliferate in response tostimulating E1E2 complexes with NS3NS4NS5a or NS3NS4NS5aNS5b peptides,with or without a core polypeptide.

HCV-specific CD4⁺ T cells can be detected by a lymphoproliferation assay(see examples). Lymphoproliferation assays measure the ability ofHCV-specific CD4⁺ T cells to proliferate in response to an E1, E2, NS3,an NS4, an NS5a, or an NS5b epitope.

Methods of Activating HCV-Specific T Cells.

The HCV proteins or polynucleotides can be used to stimulate an immuneresponse, such as to activate HCV-specific T cells either in vitro or invivo. Activation of HCV-specific T cells can be used, inter alia, toprovide model systems to optimize CTL responses to HCV and to provideprophylactic or therapeutic treatment against HCV infection. For invitro activation, proteins are preferably supplied to T cells via aplasmid or a viral vector, such as an adenovirus vector, as describedabove.

Polyclonal populations of T cells can be derived from the blood, andpreferably from peripheral lymphoid organs, such as lymph nodes, spleen,or thymus, of mammals that have been infected with an HCV. Preferredmammals include mice, chimpanzees, baboons, and humans. The HCV servesto expand the number of activated HCV-specific T cells in the mammal.The HCV-specific T cells derived from the mammal can then berestimulated in vitro by adding, e.g., HCV E1E2 and NS3NS4NS5a orNS3NS4NS5aNS5b epitopic peptides, with or without a core polypeptide, tothe T cells. The HCV-specific T cells can then be tested for, interalia, proliferation, the production of IFN-γ, and the ability to lysetarget cells displaying E1E2, NS3NS4NS5a or NS3NS4NS5aNS5b epitopes invitro.

In a lymphoproliferation assay (see examples), HCV-activated CD4⁺ Tcells proliferate when cultured with an NS3, NS4, NS5a, NS5b,NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in the absenceof an epitopic peptide. Thus, particular E1, E2, NS3, NS4, NS5a, NS5b,NS3NS4NS5a and NS3NS4NS5aNS5b epitopes that are recognized byHCV-specific CD4⁺ T cells can be identified using a lymphoproliferationassay.

Similarly, detection of IFN-γ in HCV-specific CD8⁺ T cells after invitro stimulation with the above-described HCV proteins, can be used toidentify E1, E2, E1E2, NS3, NS4, NS5a, NS5b, NS3NS4NS5a, andNS3NS4NS5aNS5b epitopes that particularly effective at stimulating CD8⁺T cells to produce IFN-γ (see examples).

Further, ⁵¹Cr release assays are useful for determining the level of CTLresponse to HCV. See Cooper et al. Immunity 10:439-449. For example,HCV-specific CD8⁺ T cells can be derived from the liver of an HCVinfected mammal. These T cells can be tested in ⁵¹Cr release assaysagainst target cells displaying, e.g., E1E2, NS3NS4NS5a and/orNS3NS4NS5aNS5b epitopes. Several target cell populations expressingdifferent E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes can beconstructed so that each target cell population displays differentepitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b. The HCV-specificCD8⁺ cells can be assayed against each of these target cell populations.The results of the ⁵¹Cr release assays can be used to determine whichepitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b are responsible forthe strongest CTL response to HCV. E1E2 complexes, NS3NS4NS5a fusionproteins or NS3NS4NS5aNS5b fusion proteins, with or without corepolypeptides, which contain the epitopes responsible for the strongestCTL response can then be constructed using the information derived fromthe ⁵¹Cr release assays.

HCV proteins as described above, or polynucleotides encoding suchproteins, can be administered to a mammal, such as a mouse, baboon,chimpanzee, or human, to stimulate an immune response, such as toactivate HCV-specific T cells in vivo. Administration can be by anymeans known in the art, including parenteral, intranasal, intramuscularor subcutaneous injection, including injection using a biologicalballistic gun (“gene gun”), as discussed above.

Preferably, injection of an HCV polynucleotide is used to activate Tcells. In addition to the practical advantages of simplicity ofconstruction and modification, injection of the polynucleotides resultsin the synthesis of a fusion protein in the host. Thus, these immunogensare presented to the host immune system with native post-translationalmodifications, structure, and conformation. The polynucleotides arepreferably injected intramuscularly to a large mammal, such as a human,at a dose of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

A composition of the invention comprising the HCV proteins orpolynucleotides is administered in a manner compatible with theparticular composition used and in an amount which is effective tostimulate an immune response, such as to activate HCV-specific T cellsas measured by, inter alia, a ⁵¹Cr release assay, a lymphoproliferationassay, or by intracellular staining for IFN-γ. The proteins and/orpolynucleotides can be administered either to a mammal which is notinfected with an HCV or can be administered to an HCV-infected mammal.The particular dosages of the polynucleotides or proteins in acomposition will depend on many factors including, but not limited tothe species, age, and general condition of the mammal to which thecomposition is administered, and the mode of administration of thecomposition. An effective amount of the composition of the invention canbe readily determined using only routine experimentation. In vitro andin vivo models described above can be employed to identify appropriatedoses. The amount of polynucleotide used in the example described belowprovides general guidance which can be used to optimize the activationof HCV-specific T cells either in vivo or in vitro. Generally, 0.5,0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg of an HCV fusion and E1 and E2polypeptides, such as an E1E2 complex, an NS3NS4NS5a or NS3NS4NS5aNS5bfusion protein or polynucleotide, with or without a core polypeptide,will be administered to a large mammal, such as a baboon, chimpanzee, orhuman. If desired, co-stimulatory molecules or adjuvants can also beprovided before, after, or together with the compositions.

Immune responses of the mammal generated by the delivery of acomposition of the invention, including activation of HCV-specific Tcells, can be enhanced by varying the dosage, route of administration,or boosting regimens. Compositions of the invention may be given in asingle dose schedule, or preferably in a multiple dose schedule in whicha primary course of vaccination includes 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand/or reenforce an immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose or doses after severalmonths.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. The accession number indicated was assignedafter successful viability testing, and the requisite fees were paid.made under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure and the Regulations thereunder (Budapest Treaty). This assuresmaintenance of viable cultures for a period of thirty (30) years fromthe date of deposit. The organisms will be made available by the ATCCunder the terms of the Budapest Treaty, which assures permanent andunrestricted availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. §1.12 with particular reference to 886 OG 638).Upon the granting of a patent, all restrictions on the availability tothe public of the deposited cultures will be irrevocably removed.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The nucleic acid sequences of these genes, as well as theamino acid sequences of the molecules encoded thereby, are incorporatedherein by reference and are controlling in the event of any conflictwith the description herein. A license may be required to make, use, orsell the deposited materials, and no such license is hereby granted.

Plasmid Deposit Date ATCC No. E1E2-809 Aug. 16, 2001 PTA-3643

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Those of skill in the art will readily appreciate that the invention maybe practiced in a variety of ways given the teaching of this disclosure.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Production of NS3NS4NS5a Polynucleotides

A polynucleotide encoding NS3NS4NS5a (approximately amino acids 1027 to2399, numbered relative to HCV-1) (also termed “NS345a” herein) or NS5a(approximately amino acids 1973 to 2399, numbered relative to HCV-1) wasisolated from an HCV. Polynucleotides encoding a methionine residue wereligated to the 5′ end of these polynucleotides and the polynucleotideswere cloned into plasmid, vaccinia virus, and adenovirus vectors.

Immunization Protocols. In one immunization protocol, mice wereimmunized with 50 μg of plasmid DNA encoding either NS5a or encoding anNS3NS4NS5a fusion protein by intramuscular injection into the tibialisanterior. A booster injection of 10⁷ pfu of vaccinia virus (VV)-NS5a(intraperitoneal) or 50 μg of plasmid control (intramuscular) wasprovided 6 weeks later.

In another immunization protocol, mice were injected intramuscularly inthe tibialis anterior with 10¹⁰ adenovirus particles encoding anNS3NS4NS5a fusion protein. An intraperitoneal booster injection of 10⁷pfu of VV-NS5a or an intramuscular booster injection of 10¹⁰ adenovirusparticles encoding NS3NS4NS5a was provided 6 weeks later.

Example 2 Immunization with DNA Encoding an NS3NS4NS5a Fusion ProteinActivates HCV-Specific CD8⁺ T Cells

⁵¹Cr Release Assay. A ⁵¹Cr release assay was used to measure the abilityof HCV-specific T cells to lyse target cells displaying an NS5a epitope.Spleen cells were pooled from the immunized animals. These cells wererestimulated in vitro for 6 days with the CTL epitopic peptide p214K9(2152-HEYPVGSQL-2160; SEQ ID NO:1) from HCV-NS5a in the presence ofIL-2. The spleen cells were then assayed for cytotoxic activity in astandard ⁵¹Cr release assay against peptide-sensitized target cells(L929) expressing class I, but not class II MHC molecules, as describedin Weiss (1980) J. Biol. Chem. 255:9912-9917. Ratios of effector (Tcells) to target (B cells) of 60:1, 20:1, and 7:1 were tested. Percentspecific lysis was calculated for each effector to target ratio.

The results of the assays are shown in Tables 1 and 2. Table 1demonstrates that immunization with plasmid DNA encoding an NS3NS4NS5afusion protein activates CD8⁺ T cells which recognize and lyse targetcells displaying an NS5a epitope. Surprisingly the NS5a polypeptide ofthe NS3NS4NS5a fusion protein was able to activate T cells even thoughthe NS5a polypeptide was present in a fusion protein.

Similarly, Table 2 demonstrates that delivery of the NS3NS4NS5a fusionprotein to mice by means of an adenovirus vector also activates CD8⁺ Tcells which recognize and lyse target cells displaying an HCV NS5aepitope. Thus, immunization with either “naked” (plasmid) DNA encodingan NS3NS4NS5a fusion protein or adenovirus vector-encoded fusion proteincan be used to activate HCV-specific T cells.

Example 3 Immunization with DNA Encoding an NS3NS4NS5a Fusion ProteinActivates HCV-Specific CD8⁺ T-Cells which Express IFN-γ

Intracellular Staining for Interferon-gamma (IFN-γ). Intracellularstaining for IFN-γ was used to identify the CD8⁺ T cells that secreteIFN-γ after in vitro stimulation with the NS5a epitope p214K9. Spleencells of individual immunized mice were restimulated in vitro eitherwith p214K9 or with a non-specific peptide for 6-12 hours in thepresence of IL-2 and monensin. The cells were then stained for surfaceCD8 and for intracellular IFN-γ and analyzed by flow cytometry. Thepercent of CD8⁺ T cells which were also positive for IFN-γ was thencalculated. The results of these assays are shown in Tables 1 and 2.Table 1 demonstrates that CD8⁺ T cells activated in response toimmunization with plasmid DNA encoding an NS3NS4NS5a fusion protein alsoexpress IFN-γ. Immunization with an NS3NS4NS5a fusion protein encoded inan adenovirus also results in CD8⁺ HCV-specific T cells which expressIFN-γ, although to a lesser extent than immunization with aplasmid-encoded NS3NS4NS5a fusion protein (Table 2).

TABLE 1 HCV-NS5a-Specific CD8+ T Cells in Mice Immunized with NS5a orNS345a DNA Intracellular ⁵¹Cr Release Assay Staining for IFN-γ PercentSpecific Percent of CD8+ T Lysis of Targets* Cells Positive for IFN-g**E:T NS5a DNA NS345a DNA NS5a DNA NS345a DNA ratio p214K9 — p214K9 —p214K9 — p214K9 — 60:1 77 5 66 6 20:1 61 4 49 2 1.74 0.26 1.18 0.40  7:129 1 29 4 *Target cells (L929) were pulsed with p214K9 or media aloneand labeled with ⁵¹Cr. **Spleen cells were cultured with p214K9 or mediaalone for 12 hours in the presence of monensin. p214K9 is a CTL epitopicpeptide (2152-HEYPVGSQL-2160, SEQ ID NO: 1) from HCV-NS5a ‘—’ refers tothe absence of peptide

TABLE 2 HCV-NS5a-Specific CD8+ T Cells Primed by Adenovirus or DNAEncoding for NS345a Intracellular ⁵¹Cr Release Assay Staining for IFN-γPercent Specific Percent of CD8+ T Lysis of Targets* Cells Positive forIFN-g** NS345a NS345a NS345a NS345a E:T Adeno DNA Adeno DNA ratio p214K9— p214K9 — p214K9 p214J p214K9 p214J 60:1 76 2 55 5 20:1 85 2 22 3 3.240.13 0.25 0.09  7:1 62 <1 10 3 *Target cells (L929) were pulsed withp214K9 or p214J and labeled with ⁵¹Cr. **Spleen cells were cultured withp214K9 or p214J for 12 hours in the presence of monensin. p214K9 is aCTL epitopic peptide (2152-HEYPVGSQL-2160, SEQ ID NO: 1) from HCV-NS5aP214J is a control peptide (10 mer) from HCV-NS5a

Example 4 Immunization with DNA Encoding an NS3NS4NS5a Fusion ProteinStimulates Proliferation of HCV-Specific CD4⁺ T Cells

Lymphoproliferation assay. Spleen cells from pooled immunized mice weredepleted of CD8⁺ T cells using magnetic beads and were cultured intriplicate with either p222D, an NS5a-epitopic peptide from HCV-NS5a(2224-AELIEANLLWRQEMG-2238; SEQ ID NO:2), or in medium alone. After 72hours, cells were pulsed with 1μ Ci per well of ³H-thymidine andharvested 6-8 hours later. Incorporation of radioactivity was measuredafter harvesting. The mean cpm was calculated.

As shown in Table 3, immunization with a plasmid-encoded NS3NS4NS5afusion protein stimulates proliferation of CD4⁺ HCV-specific T cells.Immunization with an adenovirus vector encoding the fusion protein alsoresulted in stimulated proliferation of CD4⁺ HCV-specific T cells (Table4).

TABLE 3 HCV-NS5a-Specific CD4+ T Cells in Mice Immunized with NS5a orNS345a DNA Mean CPM NS5a DNA NS345a DNA p222D media p222D media 4523 7404562 861 (x6.1) (x5.3) p222D is a CD4+ epitopic peptide (aa:2224-AELIEANLLWRQEMG-2238, SEQ ID NO: 2) from HCV-NS5a

TABLE 4 HCV-NS5-Specific CD4+ T Cells Primed by Adenovirus or DNAEncoding for NS345a Mean CPM NS345a Adeno NS345a DNA p222D media p222Dmedia 896 357 1510 385 (x2.5) (x3.9) p222D is a CD4+ epitopic peptide(aa: 2224-AELIEANLLWRQEMG-2238, SEQ ID NO: 2) from HCV-NS5a

Example 5 Efficiency of NS345a-Encoding DNA Vaccine Formulations toPrime CTLs in Mice

Mice were immunized with either 10-100 μg of plasmid DNA encoding NS345afusion protein as described in Example 1, with PLG-linked DNA encodingNS345a, described below, or with DNA encoding NS345a, delivered viaelectroporation (see, e.g., U.S. Pat. Nos. 6,132,419; 6,451,002,6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831; andInternational Publication No. WO/0045823, all of which are incorporatedherein by reference in their entireties, for this delivery technique).The immunizations were followed by a booster injection 6 weeks later of1×10⁷ pfu vaccinia virus encoding NS5a, plasmid DNA encoding NS345a orplasmid DNA encoding NS5a each as described in Example 1.

PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymers wereobtained from Boehringer Ingelheim, U.S.A. The PLG polymer used in thisstudy was RG505, which has a copolymer ratio of 50/50 and a molecularweight of 65 kDa (manufacturers data). Cationic microparticles withadsorbed DNA were prepared using a modified solvent evaporation process,essentially as described in Singh et al., Proc. Natl. Acad. Sci. USA(2000) 97:811-816. Briefly, the microparticles were prepared byemulsifying 10 ml of a 5% w/v polymer solution in methylene chloridewith 1 ml of PBS at high speed using an IKA homogenizer. The primaryemulsion was then added to 50 ml of distilled water containing cetyltrimethyl ammonium bromide (CTAB) (0.5% w/v). This resulted in theformation of a w/o/w emulsion which was stirred at 6000 rpm for 12 hoursat room temperature, allowing the methylene chloride to evaporate. Theresulting microparticles were washed twice in distilled water bycentrifugation at 10,000 g and freeze dried. Following preparation,washing and collection, DNA was adsorbed onto the microparticles byincubating 100 mg of cationic microparticles in a 1 mg/ml solution ofDNA at 4 C for 6 hours. The microparticles were then separated bycentrifugation, the pellet washed with TE buffer and the microparticleswere freeze dried.

CTL activity and IFN-γ expression were measured by ⁵¹Cr release assay orintracellular staining as described in examples 2 and 3 respectively.The results are shown in Table 5.

Results demonstrate that immunization using plasmid DNA encoding forNS345a to prime mice results in activation of CD8⁺ HCV specific T cells.

TABLE 5 Efficiency of NS345a-Encoding DNA Vaccine Formulations to PrimeCTLs in Mice ICS for IFN-gamma (% CD8+ cells that are fold IFN-g+)increase NS345a # of vs. DNA mice % # of ‘naked’ CTL Vaccines Boost MeanSdtdev P tested responding expts DNA activity? NS345a VVNS5a 1.02 1.7041 68% 10 N/A YES DNA NS345a NS345a 0.02 0.04 22 5% 5 N/A YES DNA DNANS345a NS5a 0.22 0.21 24 63% 5 N/A YES DNA DNA NS345a VVNS5a 5.00 4.36 7100% 2 4.90 YES DNA eV (electro- poration) PLGNS345a VVNS5a 2.65 2.54 6100% 2 2.60 YES DNA PLGNS345a NS5a 0.33 0.24 15 80% 3 1.50 YES DNA DNA

Example 6 Immunization Routes and Replicon Particles SINCR (DC+)Encoding for NS345a

Alphavirus replicon particles, for example, SINCR (DC+) were prepared asdescribed in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Mice were injected with 5×10⁶ IU SINCR (DC+) repliconparticles encoding for NS345a intramuscularly (IM) as described inExample 1, or subcutaneously (S/C) at the base of the tail (BoT) andfoot pad (FP), or with a combination of ⅔ of the DNA delivered via IMadministration and ⅓ via a BoT route. The immunizations were followed bya booster injection of vaccinia virus encoding NS5a as described inExample 1.

IPN-γ expression was measured by intracellular staining as described inExample 3. The results are shown in Table 6. The results demonstratethat immunization via SINCR (DC+) replicon particles encoding for NS345aby a variety of routes results in CD8+ HCV specific T cells whichexpress IFN-γ.

TABLE 6 Immunization Routes and SINCR (DC+) Replicon Particles EncodingNS345a (all mice VVNS5a challenged) ICS for IFN-gamma (% CD8+ cells thatare IFN-g+) # of mice # of % responding Vaccines Immunization Route MeanSdtdev P tested expts mice SINCR (DC+) 100% IM (ta) 1.11 0.63 3 1 100% 5× 10⁶ SINCR (DC+) 100% S/C (BoT + 0.62 0.29 3 1 100% 5 × 10⁶ FP) SINCR(DC+) 2/3 IM (ta) + 1/3 2.43 2.00 3 1 100% 5 × 10⁶ S/C (BoT)

Example 7 SINCR (DC+) Vs SINDC (LP) Replicon Particles Encoding forNS345a

Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP)were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA(1999) 96:4598-4603. Mice were immunized with 1×10³ to 1×10⁷ IU of SINCR(DC+) or SINCR (LP) replicon particles encoding for NS345a, byintramuscular injection into the tibialis anterior, followed by abooster injection of 10⁷ pfu vaccinia virus encoding NS5a at 6 weeks.

IFN-γ expression was measured by intracellular staining as described inExample 3. Administration of an increase in the number of SINCR (DC+)replicon particles encoding NS345a resulted in an increase in % of CD8+T cells expressing IFN-γ.

Example 8 Alphavirus Replicon Priming, Followed by Various BoostingRegimes

Alphavirus replicon particles, for example, SINCR (DC+) were prepared asdescribed in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Mice were primed with SINCR (DC+), 1.5×10⁶ IU repliconparticles encoding NS345a, by intramuscular injection into the tibialisanterior, followed by a booster of either 10-100 μg of plasmid DNAencoding for NS5a, 10¹⁰ adenovirus particles encoding NS345a, 1.5×10⁶ IUSINCR (DC+) replicon particles encoding NS345a, or 10⁷ pfu vacciniavirus encoding NS5a at 6 weeks.

IFN-γ expression was measured by intracellular staining as described inExample 3. The results are shown in Table 7. The results demonstratethat boosting with vaccinia virus encoding NS5a DNA results in thestrongest generation of CD8+ HCV specific T cells which express IFN-γ.Boosting with plasmid encoding NS5a DNA also results in a good response,while lesser responses are noted with adenovirus NS345a or SINCRDC+boosted animals.

TABLE 7 Alphavirus Replicon Particle Priming, Followed by VariousBoosting Regimens ICS for IFN-gamma (% CD8+ cells that are IFN-g+) # ofmice # of % responding Vaccines Boost Mean Sdtdev P tested expts miceSINCR (DC+) NS5a DNA 0.46 0.36 4 1 75% 1.5 × 10⁶ SINCR (DC+) AdenoNS345a 0.04 0.04 4 1 25% 1.5 × 10⁶ (10 × 10¹⁰) SINCR (DC+) SINCR (DC+)0.06 0.06 8 2 25% 1.5 × 10⁶ 1.5 × 10⁶ SINCR (DC+) VVNS5a (1 × 10⁷) 2.432.45 4 1 100% 1.5 × 10⁶

Example 9 Alphaviruses Expressing NS345a

Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP)were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA(1999) 96:4598-4603. Mice were immunized with 1×10² to 1×10⁶ IU SINCR(DC+) replicons encoding NS345a via a combination of delivery routes (⅔IM and ⅓ S/C) as well as by S/C alone, or with 1×10² to 1×10⁶ IU SINCR(LP) replicon particles encoding NS345a via a combination of deliveryroutes (⅔ IM and ⅓ S/C) as well as by S/C alone. The immunizations werefollowed by a booster injection of 10⁷ pfu vaccinia virus encoding NS5aat 6 weeks.

IFN-γ expression was measured by intracellular staining as described inExample 3. The results are shown in FIG. 6. The results indicateactivation of CD8+ HCV specific T cells.

Example 10 Efficiency of NS5a Encoding DNA Vaccine Formulations to PrimeCTLs in Mice

Mice were immunized with either 10-100 μg of plasmid DNA encoding NS5aas described in Example 1 or with PLG-linked DNA encoding NS5a asdescribed in Example 5. The immunizations were followed by a boosterinjection at 6 weeks of either 10-100 μg of plasmid DNA encoding forNS5a, 10¹⁰ adenovirus particles encoding NS345a, 1.5×10⁶ IU SINCR (DC+)replicon particles encoding NS345a, or 10⁷ pfu vaccinia virus encodingNS5a.

CTL activity and IFN-γ expression were measured by the methods describedin Examples 2 and 3.

The results are shown in Table 8. The results demonstrate that primingwith plasmid DNA encoding for NS5a or PLG-linked DNA encoding NS5aresults in activation of CD8+ HCV specific T cells.

TABLE 8 Efficiency of NS5a-Encoding DNA Vaccine Formulations to PrimeCTLs in Mice ICS for IFN-gamma (% CD8+ cells that are fold IFN-g+)increase # of vs. NS5a mice % # of ‘naked’ CTL Vaccines Boost MeanSdtdev P tested responding expts DNA activity? NS5a VVNS5a 1.67 1.49 8100% 3 N/A YES DNA NS5a NS5a 0.17 0.09 12 83% 3 N/A YES DNA DNA PLGNS5aNS5a 0.22 0.09 9 100% 2 1.29 YES DNA DNA NS5a AdenoNS345a 0.10 0.08 450% 1 N/A NO DNA NS5a SINCRNS345a 0.20 0.17 4 75% 1 N/A YES DNA

Example 11 Efficiency of NS345b-Encoding DNA Vaccine Formulations toPrime CTLs in Mice

Mice were immunized with 10-100 μg of plasmid DNA encoding NS34b byintramuscular injection to the tibialis anterior or with PLG linked DNAencoding NS5a as described in Example 5. The immunizations were followedby a booster injection of plasmid DNA encoding for NS5a as described inExample 1.

CTL activity and IFN-γ expression were measured by the methods describedin Examples 2 and 3.

The results are shown in Table 9. The results demonstrate that primingwith plasmid DNA encoding NS345b or PLG-linked NS345b results inactivation of CD8+ HCV specific T cells.

TABLE 9 Efficiency of NS345b-Encoding DNA Vaccine Formulations to PrimeCTLs in Mice ICS for IFN-gamma (% CD8+ cells that are fold IFN-g+)increase NS345 # of vs. DNA mice % # of ‘naked’ CTL Vaccines Boost MeanSdtdev P tested responding expts DNA activity? NS345 NS5a 0.18 0.16 1560% 3 N/A YES DNA DNA PLGNS345 NS5a 0.30 0.33 14 57% 3 1.67 YES DNA DNA

Example 12 Administration of DNA Via Separate Plasmids

Mice were immunized with 100 μg plasmid DNA encoding for NS345a or with100 μg PLG-linked DNA encoding NS345a. Additionally, separate DNAplasmids encoding NS5a, NS34a, and NS4ab (33.3 μg each) wereadministered concurrently to another group of mice. Finally, PLG-linkedDNA encoding NS5a, NS34a, and NS4ab (33.3 μg each) were administeredconcurrently to another group of mice. The immunizations were followedby a booster injection of 1×10⁷ pfu vaccinia virus encoding NS5a, 6weeks post first immunization.

IFN-γ expression was measured by the method described in Example 3. Theresults are shown in FIG. 7. The results demonstrate a particularlyvigorous response in the activation of CD8+ HCV specific T cells whenthe DNA is broken down into smaller sub units and linked to PLG.

Example 13 Immunogenicity of NS345Core₁₂₁-ISCOMS in Mice

Groups of 10 C57 black mice were immunized IM at 0, 21 and 60 days withthe formulations shown in Table 10. The NS345Core₁₂₁-PLGdss groupreceived a vaccine dose of 50 μl in each leg whereas the other vaccinegroups received a vaccine dose of 50 μl in one leg.

NS345Core₁₂₁-ISCOMS were comprised of amino acids 1242 to 3011 and 1-121and the HCV polyprotein, numbered relative to HCV-1 and were adsorbed toISCOMS with a ratio of protein to QH of approximately 8:1, usingstandard techniques. See, e.g., International Publication No. WO01/37869A, incorporated herein by reference in its entirety.

Core-ISCOMS including an HCV core protein fragment from the regionspanning amino acid positions 1-191 of the HCV polyprotein, numberedrelative to HCV-1, with a ratio of protein to QH of 1:1, were producedusing standard techniques. See, e.g., International Publication No. WO01/37869A, incorporated herein by reference in its entirety.

NS345Core₁₂₁ was formulated in 0.1% SDS in PBS and contained DTT.Protein was diluted in PBS and mixed 1:1 with MF59 (see, Ott et al.,“MF59—Design and Evaluation of a Safe and Potent Adjuvant for HumanVaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell,M. F. and Newman, M. J. eds.) Plenum Press, New York (1995) pp. 277-296;and U.S. Pat. No. 6,299,884, incorporated herein by reference in itsentirety) prior to immunization.

For NS345Core₁₂₁-PLGdss, PLG microparticles produced as described abovewere treated with 3-(trimethylsilyl)-1-propanesulfonic acid (DSS) toenhance adsorption of antigen. DSS is commercially available from, e.g.,Sigma Chemical Co., St. Louis, Mo. NS345Core₁₂₁ was adsorbed theretousing standard techniques (see, International Publication No. WO00/050006). The NS345Core₁₂₁-PLGdss was mixed with MF59 prior toimmunization.

As shown in Table 10, NS345Core₁₂₁-ISCOMS produced antibody responseonly to NS5 in immunized C57 black mice. Higher levels of antibodies toNS5 were produced in mice immunized with NS345Core₁₂₁ adjuvanted withMF59, however no antibody response to core, NS3 or NS4 was produced withthis adjuvant either.

Mice immunized with Core-ISCOMS produced antibodies to core. Incontrast, NS345Core₁₂₁-PLGdss immunized mice produced significantlyhigher antibodies to NS5 than NS345Core₁₂₁-ISCOMS. In addition,NS345Core₁₂₁-PLGdss immunized mice produced antibodies to NS3 and someantibody response to core, but no antibodies to NS4.

TABLE 10 Immunogenicity of NS345Core₁₂₁-Iscoms in Mice. Geometric meanEIA antibody titers to core and nonstructural proteins are shown. Thenumber of responding mice per group are also listed. Anti-C33C Anti-C100Anti-Core (NS3) (NS4) Anti-NS5 IM Protein Dose Antibody AntibodyAntibody Antibody Vaccine (μg)^(a) EIA GMT EIA GMT EIA GMT EIA GMTNS345Core₁₂₁ 6.0, 6.0, 6.0^(b) <10 <10 <10  31 ISCOMS (0/10) (0/10)(0/10) (7/10) Core- 6.0, 6.0, 6.0^(c) 188 <10 <10 <10 ISCOMS (9/10)(1/10) (0/10) (2/10) NS345Core₁₂₁ 6.0, 6.0, 6.0 <10 <10 <10 279 MF59(2/9)  (1/9)  (0/9)  (9/)  NS345Core₁₂₁ 10, 10, 10  5  50 <10 419 PLG-(6/10) (9/10) (2/10) (9/9)  dss/MF59 ^(a)Groups of 10 C57 black micewere immunized IM at 0, 21 and 60 days. Serum was obtained after thelast immunization. The NS345 Core₁₂₁-PLGdss group received vaccine doseof 50 μl in each leg whereas the other vaccine groups received vaccinedose of 50 μl in one leg. ^(b)The ratio of protein to QH wasapproximately 8:1. ^(c)The ratio of protein to QH was approximately 1:1.

Example 14 Immunogenicity of Different Formulations of NS345Core₁₂₁ orNS345 in Mice

Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days withthe formulations shown in Tables 11 and 12. For the studies shown inTable 11, the NS345 and NS345Core₁₂₁ protein concentration was 10 μg perdose, and for those in Table 12, the concentration was 5 μg per dose.

For PLG-NS345 (amino acids 1242 to 3011 of the HCV polyprotein) andPLG-NS345Core₁₂₁ (amino acids 1242-3011 and 1-121 of the HCVpolyprotein), PLG microparticles were prepared and NS345 or NS345Core₁₂₁were adsorbed thereto using standard techniques, as described above.

For PLG-NS345+PLG-CTAB-E1E2 DNA, PLG microparticles were prepared andNS345 was adsorbed to the microparticles as described above. E1E2 DNAwas produced as follows. Mammalian expression plasmid pMH-E1E2-809 (FIG.4, ATCC Deposit No. PTA-3643) encodes an E1E2 fusion protein whichincludes amino acids 192-809 of HCV 1a (see, Choo et al., Proc. Natl.Acad. Sci. USA (1991) 88:2451-2455). Chinese Hamster Ovary (CHO) cellswere used for expression of the HCV E1E2 sequence from pMH-E1E2-809. Inparticular, CHO DG44 cells were used. These cells, described by Uraub etal., Proc. Natl. Acad. Sci. USA (1980) 77:4216-4220, were derived fromCHO K−1 cells and were made dihydrofolate reductase (dhfr) deficient byvirtue of a double deletion in the dhfr gene. DG44 cells weretransfected with pMH-E1E2-809. The transfected cells were grown inselective medium such that only those cells expressing the dhfr genecould grow (Sambrook et al., supra). Isolated CHO colonies were picked(˜800 colonies) into individual wells of a 96-well plate. From theoriginal 96-well plates, replicates were made to perform expressionexperiments. The replicate plates were grown until the cells made aconfluent monolayer. The cells were fixed to the wells of the plate andpermeablized using cold methanol. Anti-E1 and anti-E2 antibodies, 3D5C3and 3E5-1 respectively, were used to probe the fixed cells. After addingan anti-mouse HRP conjugate, followed by substrate, the cell lines withthe highest expression were determined. The highest expressing celllines were then expanded to 24-well cluster plates. The assay forexpression was repeated, and again, the highest expressing cell lineswere expanded to wells of greater volume. This was repeated until thehighest expressing cell lines were expanded from 6-well plates intotissue culture flasks. At this point there was sufficient quantity ofcells to allow accurate count and harvest of the cells, and quantitativeexpression assays were done. An ELISA was performed on the cell extract,to determine high expressors.

To produce the PLG-CTAB-E1E2 DNA, PLG microparticles were treated withCTAB as described above (see, International Publication No. WO00/050006).

For PLG-NS345Core₁₂₁+E1E2 DNA PLG-NS345Core₁₂₁ and E1E2 DNA wereproduced as described above.

For PLG-NS345 or PLG-NS345Core₁₂₁+MF59, PLG-NS345 or PLG-NS345Core₁₂₁,was combined with MF59 as described above.

For PLG-NS345 or PLG-NS345Core₁₂₁+CTAB-CpG, NS345 or NS345Core₁₂₁ wasadsorbed to PLG as described above. The CpG molecule used was5′-TCCATGACGTTCCTGACGTT-3′ and this was treated with CTAB, as describedabove.

For PLG-NS345 or PLG-NS345Core₁₂₁+QS21, the saponin adjuvant QS21 wascombined with the PLG-HCV proteins.

For PLG-NS345 or PLG-NS345Core₁₂₁+CTAB-CpG+MF59, the various components,as described above, were combined.

The remaining adjuvants used in the studies and shown in the tables areself-explanatory.

The results of these studies are shown in Tables 11 and 12. As can beseen in Table 11, none of the formulations produced antibody responsesto core, NS3 or NS4 antigens. However, PLG-NS345+CTAB-CPG in MF59produced the highest antibody titers to NS5. PLG-NS345Core₁₂₁+QS21,PLG-NS345+CTAB-CPG, PLG-NS345Core₁₂₁+CTAB-CPG, and PLG-NS345+QS21produced moderate antibody titers to NS5. The other formulationsproduced very low antibody titers to NS5.

As can be seen in Table 12, NS345Core₁₂₁/MF59/MPL andNS345Core₁₂₁/MF59/CpG formulations produced very high antibody titers toNS345Core₁₂₁, NS345Core₁₂₁/MF59, NS345/MF59/CpG, andNS345Core₁₂₁/MF59/Cho1/QS21 formulations produced moderate antibodytiters to NS345Core₁₂₁. The other formulations produced very low or noantibody titers to NS345Core₁₂₁.

TABLE 11 Immunogenicity of different formulations of HCV NS345Core₁₂₁ orNS345 in Mice. Geometric mean EIA antibody titers to core andnonstructural proteins are shown. Anti-Core Anti-C33C Anti-C100 Anti-NS5Antibody EIA (NS3) Antibody (NS4) Antibody Antibody EIA Vaccine^(a) GMTEIA GMT EIA GMT GMT PLG-NS345 <10 <10 <10 10 PLG- <10 <10 <10 15NS345Core₁₂₁ PLG- <10 11 <10 23 NS345 + PLG- CTAB-E1E2 DNA PLG- <10 <10<10 20 NS345Core₁₂₁ + E1E2 DNA PLG-NS345 + <10 <10 <10 70 MF59 PLG- <10<10 <10 26 NS345Core₁₂₁ + MF59 PLG-NS345 + <10 <10 <10 350 CTAB-CPG PLG-<10 <10 <10 271 NS345Core₁₂₁ + CTAB-CPG PLG-NS345 + <10 <10 <10 201 QS21PLG- <10 <10 <10 505 NS345Core₁₂₁ + QS21 PLG-NS345 + <10 <10 <10 1471CTAB-CPG + MF59 PLG- <10 <10 <10 63 NS345Core₁₂₁ + CTAB + MF59^(a)Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days.Serum was obtained after the last immunization. The NS345 orNS345Core₁₂₁ protein concentration was 10 μg per dose.

TABLE 12 Immunogenicity of different formulations of HCV NS345Core₁₂₁ orHCV NS345 in Mice. Geometric mean EIA antibody titers to NS345Core₁₂₁protein are shown. Anti-NS345Core₁₂₁ Vaccine^(a) Antibody EIA GMTNS345Core₁₂₁/MF59 328 NS345Core₁₂₁/MF59/CpG 7,926 NS34a + NS5B +Core/MF59 12 NS34a + NS5B + Core/MF59/CpG 5 PLG-NS345Core₁₂₁/MF59 <10PLG-NS345Core₁₂₁/MF59/CpG <10 PLG-NS345Core₁₂₁/PLG-CpG 9NS345Core₁₂₁/alum phosphate 34 NS345Core₁₂₁/alum phosphate//CpG 950NS345/MF59/CpG 511 PLG-NS345/PLG/CpG 117 NS345Core₁₂₁/MF59/MPL 10,292NS345Core₁₂₁/MF59/Chol/QS21 698 NS345Core₁₂₁/Alum phosphate/MPL 23^(a)Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days.Serum was obtained after the last immunization. The NS345 orNS345Core₁₂₁protein concentration was 5 μg per dose.

Example 15 Lymphoproliferative Response of if Different Formulations ofNS345Core₁₂₁ or NS34A+NS35B+Core in Mice

Groups of 8 C57 black mice were immunized IM at 0, 30 and 60 days withthe formulations shown in Table 13 and are as described above. Spleenswere obtained after the last immunization. The NS345Core₁₂₁ proteinconcentration was 25 μg per dose. The NS34a, NS5b and core doses were 3μg each.

The results of this study are shown in Table 13. As can be seen,NS345Core₁₂₁/Alum/CpG, PLG-NS345Core₁₂₁/PLG/CpG,NS34a+NS5B+Core/MF59/CpG and PLG-NS345Core₁₂₁/MF59/CpG formulationsdemonstrated strong LPA responses to NS5, NS34 and core antigens. TheNS345Core₁₂₁/MF59 formulation also produced a strong LPA response to NS5and NS34. Core was not tested. Moderate LPA responses were observed toNS5, NS34 and Core antigens with PLG-NS345Core₁₂₁/MF59 andNS34a+NS5B+Core/MF59 formulations. The NS345Core₁₂₁/MF59/CpG formulationmay not have been administered properly in that no LPA response wasobserved in this experiment. In a subsequent experiment as shown inTable 14, an LPA was observed to this formulation.

Groups of 8 C57 black mice were immunized once IM with the formulationsshown in Table 14, produced as described above. Draining lymph nodeswere obtained. The NS345Core₁₂₁ protein concentration was 25 μg perdose.

The results of this study are shown in Table 14. As can be seen in Table14, all the formulations tested produced a strong LPA response to NS5,NS34 and Core as well as the NS345Core₁₂₁.

TABLE 13 Lymphoproliferative response of different formulations of HCVNS345Core₁₂₁ or NS34A + NS5B + Core in Mice. LPA responses (cpm) to coreand nonstructural proteins are shown. The number of mice in each groupresponding is also indicated in parentheses. HIV-2 env SOD-C200SOD-C22-3 (background Vaccine^(a) SOD-NS5 (NS34) (Core) control)NS345Core₁₂₁/MF59 2250 1800 ND 144 (6/8) (4/8) NS345Core₁₂₁/MF59/CpG  80 80 ND 138 PLG-NS345Core₁₂₁/MF59  560  120 510 93 (2/8) (2/8) (2/8) PLG-1600 1500 620 75 NS345Core₁₂₁/MF59/CpG (6/8) (6/8) (8/8) NS34a + NS5B +Core/MF59  564  710 265 76 (8/8) (8/8) (8/8) NS34a + NS5B + 1523  885446 67 Core/MF59/CpG (8/8) (8/8) (6/8) PLG-NS345Core₁₂₁/PLG/CpG 36752860 370 88 (8/8) (8/8) (8/8) NS345Core₁₂₁/Alum/CpG 8450 7940 1040  82(8/8) (8/8) (6/8) ^(a)Groups of 8 C57 black mice were immunized IM at 0,30 and 60 days. Spleens were obtained after the last immunization. TheNS345Core₁₂₁ protein concentration was 25 μg per dose. The NS34a, NS5Band Core doses were 3 μg each.

TABLE 14 Lymphoproliferative response of different formulations of HCVNS345Core₁₂₁ in Mice. The LPA responses (cpm) form an average of threeconsecutive experiments to core and nonstructural proteins are shown.HIV-2 env SOD-C200 SOD-C22-3 (background Vaccine^(a) SOD-NS5 (NS34)(Core) NS345Core₁₂₁ control) NS345Core₁₂₁/MF59 8900 8300 3000 20900 890NS345Core₁₂₁/MF59/CpG 1890 1628 1200 20100 623 PLG-NS345Core₁₂₁/MF5910700 12900 1800 23700 818 PLG- 3600 4690 1660 27300 911NS345Core₁₂₁/MF59/CpG PLG-NS345Core₁₂₁ 4500 4660 760 18150 315PLG-NS345Core₁₂₁/PLG/CpG 7750 5300 1250 22980 450 NS345Core₁₂₁/Alum 30503725 744 11300 390 NS345Core₁₂₁/Alum/CpG 4130 4670 600 20660 480^(a)Groups of 8 C57 black mice were immunized once IM. Draining lymphnodes were obtained. The NS345Core₁₂₁ protein concentration was 25 μgper dose.

Example 16 Immunogenicity of Recombinant HCV Protein Vaccines Adjuvantedwith ISCOMS in Rhesus Macaques

The safety and immunogenicity of HCV proteins completed with theadjuvant, Iscomatrix, was studied in Rhesus macaques. Three groups madeup of four animals each were immunized IM as detailed below at week 0, 4and 8 weeks. Vaccines were prepared as described above. The ISCOMS usedlacked QH-A.

Group Number n Vaccine Delivery 1 4 Core-ISCOM  0.5 ml R Leg (50 μg in 1ml)  0.5 ml L Leg 2 4 NS345Core₁₂₁-ISCOM  0.5 ml R Leg (1 mg in 1 ml) 0.5 ml L Leg 3 4 Core-ISCOM  0.5 ml Core-ISCOM R Leg (25 μg in 0.5 ml)and 0.35 ml NS5b-ISCOM L Leg NS5b-ISCOM (50 μg in 0.35 ml)Bleeds occurred as follows and immunogenicity was determined by CTLassays, lymphoproliferation assays, FACS analysis and antibody responsea previously described (Palakos, et al. (2001) J. of Immunology166:3589).

Week Bleed date Immunized −10 −1 X 0 X 2 X 4 X 6 X 8 X 10 X

The immunogenicity of the different HCV recombinant protein vaccines isshown in Tables 15-17.

TABLE 15 The Immunogenicity of HCV Core-ISCOMS vaccine two weeks post2^(nd) immunization and post 3^(rd) immunization as assessed by CTLassays, CD8+ FACS analysis, LPA stimulation index and CD4+ FACS analysisCD8+ ICS (CTL) CD4+ ICS (LPA SI) Macaque # C NS3 NS4 NS5a NS5b C NS3 NS4NS5a NS5b 2 weeks post 3° X020 −(−) +(−) N001 −(−) +(−) N086 −(−) +(−)X010 −(−) +(11) 2 weeks post 2° X020 −(−) +/−(−) N001 −(−) −(8) N086−(−) +(−) X010 −(−) +/− (12)

TABLE 16 The Immunogenicity of HCV NS345Core₁₂₁-ISCOMS vaccine two weekspost 2^(nd) immunization and post 3^(rd) immunization as assessed by CTLassays, CD8+ FACS analysis, LPA stimulation index and CD4+ FACS analysisCD8+ ICS (CTL) CD4+ ICS (LPA SI) Macaque # C NS3 NS4 NS5a NS5b C NS3 NS4NS5a NS5b 2 weeks post 3° X016 −(−) +(+) −(−) −(−) −(−) −(−) +(−) −(−)+/−(−) +/−(−) X008 −(−) −(−) −(−) −(−) −(−) −(−) −(−) −(−) −(5) −(−)X021 −(−) −(−) −(−) −(−) −(−) −(−) −(−) −(−) −(−) −(−) X023 +/− (−) −(−)−(−) +/−(−) −(−) −(−) +/−(−) −(−) −(−) −(−) 2 weeks post 2° X016 − (−)+(+) −(−) +(+) +(+) −(−) +(−) +/−(−) +(−) +(−) X008 +(−) +(+) −(−) +(+)+(+) −(−) +(−) −(−) +−(−) +(−) X021 −(−) −(−) +/−(−) −(−) +/−(−) −(−)−(−) +/−(−) −(−) −(−) X023 +/−(+) +(+) −(−) +/−(−) +(−) −(−) +(−) −(−)−(−) 7

TABLE 17 The Immunogenicity of HCV Core-ISCOMS + NS5b-ISCOMS vaccine twoweeks post 2^(nd) immunization and post 3^(rd) immunization as assessedby CTL assays, CD8+ FACS analysis, LPA stimulation index and CD4+ FACSanalysis CD8+ ICS (CTL) CD4+ ICS (LPA SI) Macaque # C NS3 NS4 NS5a NS5bC NS3 NS4 NS5a NS5b 2 weeks post 3° X022 +(−) −(−) −(8) +/−(11) X014−(−) −(−) +(6) +(11) N154 −(−) −(−) −(−) +(−) N173 −(−) −(−) −(−) +/−(−)2 weeks post 2° X022 −(−) −(+) −(−) −(−) X014 +(−) +/−(−) −(−) +(−) N154−(−) −(−) −(6) +(8) N173 −(−) −(−) +/−(−) +(6)

As can be seen in Table 15, the HCV Core-ISCOM vaccine produced no CTLpositive responses in any of the 4 immunized macaques after the secondor third immunizations. No positive CD8 γ-interferon and/or TNF-αintracellular staining was also observed, although backgrounds were highin these particular arrays. At least two of four macaques produced astrong LPA response after the second immunizations, but only oneremained positive after the third immunization. Two of four macaquesproduced positive CD4 intracellular staining after the secondimmunization and four of four after the third immunization.

As shown in Table 16, the HCV NS345Core₁₂₁-ISCOM vaccine after thesecond immunization produced CTL positive responses to peptide poolsrepresenting two or more HCV proteins in three of four macaques (two ofthese macaques had responses to peptide pools from NS3, NS5a and NS5b,one to peptide pools from core and NS3). CD8 positive γ-interferonand/or TNF-α intracellular staining to peptide pools representing two ormore HCV proteins was positive in at least three of four macaques. Oneof four macaques produced a strong LPA response. At least three of fourmacaques produced CD4 positive intracellular staining to two or more HCVproteins. After the third immunization, only one of four macaques had apositive CTL response, CD8 positive intracellular staining and C04positive intracellular staining. One other macaque had a positive LPAresponse and weak CD8+CD4 intracellular staining, This decline inimmunogenicity was likely due to instability of the vaccine formulation(see below).

As shown in Table 17, the HCV Core-ISCOM+NS5b-ISCOM vaccine produced aCTL positive response to NS5b in one of the 4 immunized macaques afterthe second immunization which did not remain positive after the thirdimmunization. CD8 positive intracellular positive staining was observedin one of four animals post second. Two of four macaques produced astrong LPA response after the second immunization which did not remainpositive after the third immunization. Two other macaques did develop astrong LPA response after the third immunization. Three or fourdeveloped positive CD4 intracellular staining. One developed positiveCD8 intracellular staining.

Three weeks after the third immunization, it was noted that the physicalappearance of the polyprotein vaccine solution was visibly turbid. Thecore vaccine also was turbid but less so. The Core-NS5 vaccine was alsoslightly turbid. Analysis of this turbidity in the polyproteinformulation indicated that the ISCOM particles had precipitated intolarge aggregates. These aggregates could be dispersed by vortexing with0.1% TWEEN 80 detergent. It is probable that this change in theformulation of the vaccine occurred before the last immunization. Thisobserved change in appearance of the vaccines may have affected theirimmunogenicity as cellular immune results declined in all threevaccines.

The immunogenicity of HCV Core-ISCOMS, NS345Core₁₂₁-ISCOMS andCore-ISCOMS+NS5b-ISCOMS as assessed by EIA antibody response is shown inTable 18. As can be seen, all three vaccines produced an antibodyresponse by the third immunization to their corresponding HCV proteins,except for the NS345Core₁₂₁-ISCOM vaccine. The NS345Core₁₂₁-ISCOMvaccine produced antibody responses to NS3, NS4 and a very strongantibody response to NS5, but no antibody response to HCV core.

TABLE 18 The immunogenicity of HCV Core-ISCOMS, NS345Core₁₂₁-ISCOMS,Core- ISOCMS + NS5b-ISCOMS vaccine two weeks post 2^(nd) immunizationand post 3^(rd) immunization as assessed by EIA antibody response to HCVproteins. Anti-Core EIA Anti-NS3 EIA Anti-NS4 EIA Anti-NS5 EIA VaccineAntibody Titer Antibody Titer Antibody Titer Antibody Titer Macaque #Post 2^(nd) Post 3^(rd) Post 2^(nd) Post 3^(rd) Post 2^(nd) Post 3^(rd)Post 2^(nd) Post 3^(rd) Core- ISCOM X020 66 226 N001 87 46 N086 363 396X010 108 137 NS345 Core121/ ISCOM X016 <10 <10 <10 554 56 68 3,590 3,405X008 <10 <10 66 995 14 44 2,109 3,213 X021 <10 <10 128 6,330 41 2047,213 8,083 X023 <10 <10 <10 3,910 64 64 1,243 4,704 Core- ISCOM + NS5b-ISCOM X022 <10 18 <10 134 X014 <10 13 <10 693 N154 542 554 <10 272 N17328 78 <10 258

Example 17 Immunization of Chimpanzees with Recombinant HCV Protein andDNA Vaccines

Five groups of five chimps each were immunized IM at 0, 0.7, 2 and 5months with the formulations presented below. Blood was collected atweek 0, two weeks subsequent to the second immunization, two weeksfollowing the third immunization and two weeks after the fourthimmunization.

Formulation 1: 20 μg E1E2 polypeptide+MF59+500 μg CpG (produced asdescribed above);

Formulation 2: 1 mg NS345Core₁₂₁-ISCOM (produced as described above);

Formulation 3: 6 mg each of CTAB-PLG-E1E2 (bp 574-2427, encoding aminoacids 192-809 of the HCV polyprotein, numbered relative to HCV-1);CTAB-PLG-NS34a (bp 3079-5133, encoding amino acids 1027-1711 of the HCVpolyprotein, numbered relative to HCV-1); CTAB-PLG-NS34ab (bp 4972-5916,encoding amino acids 1658-1972 of the HCV polyprotein, numbered relativeto HCV-1); CTAB-PLG-NS5a (bp 5917-7260, encoding amino acids 1973-2420of the HCV polyprotein, numbered relative to HCV-1);

Formulation 4: 6 mg each of E1E2 DNA, NS34a DNA, NS34ab DNA and NS5aDNA, having the same coordinates as described above, delivered withoutPLG via electroporation (see, e.g., U.S. Pat. Nos. 6,132,419; 6,451,002,6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831; andInternational Publication No. WO/0045823, all of which are incorporatedherein by reference in their entireties, for this delivery technique).Results are shown in FIGS. 8-10.

As can be seen, in FIG. 8, all vaccines were capable of priming CD4+ andCD8+ cells specific to HCV. Thus, all vaccines were successful atinducing a T cell response to HCV. Determination of the results for thePLG-DNA from formulation 3 at two weeks subsequent to the fourthvaccination is in progress.

As shown in FIGS. 9 and 10, multiple T cell specificities were inducedby the two vaccines. Both vaccines primed T-cells specific for multipleT cell epitopes.

As can be seen in Tables 19 and 20, E1E2 adjuvanted with MF59 primedanti-E1E2 titers. CpG enhanced anti-E1E2 responses as well as TH1responses and the ISCOM and the two DNA vaccines were capable of primingCD4+ and CD8+ T cell responses to HCV.

TABLE 19 Anti-E1E2 EIA antibody titers in chimps immunized withElectroporated DNA E1E2NS345a or PLG DNA E1E2NS345 Vaccine Chimp Pre1^(st) Post 2^(nd) Post 3^(rd) Post 4th Electroporated DNA 4X0330 — — —9 E1E2-NS34A- 4X0335 — — — 10 NS4AB-NS5A^(a) 4X0348^(c) 10 457 198 504X0354^(d) 206 1,261 1,197 207 4X0368^(c) 245 1,426 1,267 358 PLG DNA4X0238 — — 30 10 E1E2-NS34A- 4X0239 — 104 309 — NS4AB-NS5A^(b) 4X0250 —— 12 — 4X0278 — — 29 — 4X0288 — — 12 — ^(a)Electroporated IM with 1.5 mgof each plasmid at 0, 0.7, 2 and 5 months. Bleeds were taken 14 daysafter each immunization. ^(b)IM immunization with 1.5 mg of each PLGplasmid at 0, 0.7, 2 and 6 months. Bleeds were taken 14 days after eachimmunization. ^(c)Prior E2 immunization ^(d)Prior E1E2 immunization

TABLE 20 Immunogenicity in chimps of low dose (20 μg) HCV E1E2 antigenusing MF59 or MF59 combined with CpG as adjuvants (2 wks post 3^(rd))E1E2EIA E1E2 EIA CD4+ Vaccine^(a) Chimp Ab Titer Ab GMT (ICS) E1E2/ 4 ×0419 84 − MF59 4 × 0420 101 − 4 × 0431 131   261 − 4 × 0371 421 − 4 ×0372 2,580 +/− P = 0.029^(b) E1E2/ 4 × 0410 8,835 − MF59/CpG 4 × 04262,713 − 4 × 0365 3,201 2,713 + 4 × 0367 510 − 4 × 0346 1,238 ++^(a)Chimps immunized IM at 0, 1 and 6 mos with 20 μg of E1E2 antigenusing MF59 with or without 500 μg of CpG. Serum samples were obtained 14days after last immunization. ^(b)Chimps immunized with E1E2 using CpGcombined with MF59 as adjuvant produced significantly higher (P < 0.05)levels of E1E2 EIA antibody than chimpanzees with E1E2 using MF59 alone.

Thus, HCV polypeptides and polynucleotides, either alone or as fusions,to stimulate cell-mediated immune responses, are disclosed. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

1. A composition comprising: (a) a fusion protein comprising HCVpolypeptides, wherein the HCV polypeptides consist of an NS3, an NS4, anNS5a, and NS5b polypeptide of a hepatitis C virus (HCV) and optionally acore polypeptide; and (b) a polynucleotide encoding an HCV E1E2 complex,wherein the composition stimulates an immune response to HCV.
 2. Thecomposition of claim 1, wherein at least one of the HCV polypeptides isderived from a different strain of HCV than the other HCV polypeptides.3. The composition of claim 1, further comprising a pharmaceuticallyacceptable excipient.
 4. The composition of claim 3, further comprisingan adjuvant.
 5. The composition of claim 3, further comprising a CpGoligonucleotide.
 6. The composition of claim 3, wherein said fusionprotein is adsorbed to or entrapped within a microparticle or ISCOM. 7.A composition comprising a hepatitis C virus (HCV) polynucleotideencoding an HCV E1E2 complex, HCV polypeptides, and a pharmaceuticallyacceptable excipient, wherein the HCV polypeptides consist of: (a) anisolated and purified NS3 polypeptide; (b) an isolated and purified NS4;(c) an isolated and purified NS5a polypeptide; (d) an isolated andpurified NS5b polypeptide; and optionally, (e) an isolated and purifiedcore polypeptide, wherein the composition stimulates an immune responseto HCV.
 8. The composition of claim 7, further comprising an adjuvant.9. The composition of claim 7, further comprising a CpG oligonucleotide.10. The composition of claim 7, wherein one or more of said HCVpolypeptides is adsorbed to or entrapped within a microparticle orISCOM.
 11. A method of activating T cells of a vertebrate subject whichrecognize an epitope of an HCV polypeptide, comprising the step of:administering the composition of claim 1 to said vertebrate subject,whereby a population of activated T cells recognizes an epitope of theNS3, NS4, NS5a, NS5b and/or core polypeptides.
 12. A method ofactivating T cells of a vertebrate subject which recognize an epitope ofan HCV polypeptide, comprising the step of: administering thecomposition of claim 4 to said vertebrate subject, whereby a populationof activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5band/or core polypeptides.
 13. A method of activating T cells of avertebrate subject which recognize an epitope of an HCV polypeptide,comprising the step of: administering the composition of claim 5 to saidvertebrate subject, whereby a population of activated T cells recognizesan epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
 14. Amethod of activating T cells of a vertebrate subject which recognize anepitope of an HCV polypeptide, comprising the step of: administering thecomposition of claim 6 to said vertebrate subject, whereby a populationof activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5band/or core polypeptides.
 15. A method of activating T cells of avertebrate subject which recognize an epitope of an HCV polypeptide,comprising the step of: administering the composition of claim 7 to saidvertebrate subject, whereby a population of activated T cells recognizesan epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.